US20130129668A1 - Diagnosis and treatment of arthritis using epigenetics - Google Patents

Diagnosis and treatment of arthritis using epigenetics Download PDF

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Publication number: US20130129668A1
Authority: US; United States
Prior art keywords: osteoarthritis; loci; rheumatoid arthritis; methylation; genes
Prior art date: 2011-09-01
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.): Abandoned

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US13/573,233

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English (en)

Inventor

Gary Firestein

Kazuhisa Nakano

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University of California San Diego UCSD

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University of California San Diego UCSD

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2011-09-01

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2012-08-31

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2013-05-23

2012-08-31 Application filed by University of California San Diego UCSD filed Critical University of California San Diego UCSD

2012-08-31 Priority to US13/573,233 priority Critical patent/US20130129668A1/en

2013-05-23 Publication of US20130129668A1 publication Critical patent/US20130129668A1/en

Status Abandoned legal-status Critical Current

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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/154—Methylation markers

Definitions

Embodiments of the present invention include methods, compositions and kits for evaluating a diagnosis, prognosis, or response to treatment of a subject with a disorder such as rheumatoid arthritis or osteoarthritis. Some embodiments include identifying a therapeutic agent for treating a disorder such as rheumatoid arthritis or osteoarthritis.
Rheumatoid arthritis is an immune-mediated disease marked by symmetric inflammation in diarthrodial joints and destruction of the extracellular matrix.
Genomics has rapidly advanced our understanding of susceptibility and severity of RA, and many associated polymorphisms in key genes have been described. However, identical twins have a concordance rate of only 12-15% suggesting that other influences can affect either the onset or progression of disease, such as epigenetic regulation of gene expression.
Fibroblast-like synoviocytes which form the synovial intimal lining, play an integral role by producing key cytokines (e.g., IL-6), small molecule mediators (e.g., prostanoids), and proteases (e.g., metalloproteinases). While osteoclasts are the primary effectors of bone erosions in arthritis, FLS are responsible for cartilage damage by virtue of their ability to adhere to and invade cartilage extracellular matrix.
This capacity requires homotypic aggregation mediated by the adhesion molecule cadherin-11, which directs intimal lining formation and supports an invasive phenotype (Kiener H P et al. Cadherin 11 promotes invasive behavior of fibroblast-like synoviocytes. Arthritis Rheum. 2009 May; 60(5):1305-10).
a well-defined relationship has long been recognized between synovial tissue histology and function and peripheral blood cell characteristics in diseases like RA, most likely because the circulating cells actively traffic between the synovium, lymph nodes, and peripheral blood (Malone D G et al. Immune function in severe, active rheumatoid arthritis.
Some embodiments of the methods, compositions and kits provided herein include a method for diagnosing rheumatoid arthritis or osteoarthritis, determining a rheumatoid arthritis or osteoarthritis prognosis, or determining or predicting a response to treatment for rheumatoid arthritis or osteoarthritis in a subject comprising determining whether at least 2 nucleic acid loci or at least 2 genes in a sample from said subject have methylation states indicative of rheumatoid arthritis, osteoarthritis, a rheumatoid arthritis or osteoarthritis prognosis, or a response to treatment for rheumatoid arthritis or osteoarthritis.
Some embodiments also include comprising comparing the methylation states of the at least 2 loci or at least 2 genes in the sample from said subject with the methylation states of the loci or genes in normal tissue, tissue from a subject without said known rheumatoid arthritis or osteoarthritis prognosis, or tissue from a subject without said known response to treatment for rheumatoid arthritis or osteoarthritis.
an increase or decrease in the extent of methylation of at least 2 loci or at least 2 genes compared to the extent of methylation of the loci or genes in normal tissue, tissue from a subject without said known rheumatoid arthritis or osteoarthritis prognosis, or tissue from a subject without said known response to treatment for rheumatoid arthritis or osteoarthritis is indicative of the presence or absence of rheumatoid arthritis, osteoarthritis, a rheumatoid arthritis or osteoarthritis prognosis, or response to treatment for rheumatoid arthritis or osteoarthritis for the subject.
the methylation states of said at least 2 loci or at least 2 genes are determined in fibroblasts from said subject. In some embodiments, the methylation state of said at least 2 loci or at least 2 genes is determined in synoviocytes from said subject. In some embodiments, the methylation states of said at least 2 loci or at least 2 genes are determined in fibroblast-like synoviocytes from said subject. In some embodiments, the methylation states of said at least 2 loci or at least 2 genes are determined in rheumatoid arthritis fibroblast-like synoviocytes from said subject. In some embodiments, the methylation states of said at least 2 loci or at least 2 genes are determined in osteoarthritis fibroblast-like synoviocytes from said subject.
the methylation states of said at least 2 loci or at least 2 genes are determined in macrophage from said subject.
the methylation states of said at least 2 loci or at least 2 genes are determined in peripheral blood cells from said subject.
the peripheral blood cell is selected from the group consisting of white blood cell, neutrophil, eosinophil, basophil, lymphocyte, B cell, plasma cell, T cell, natural killer cell, monocyte, and dendritic cell.
the sample is selected from the group consisting of a tissue sample, a frozen tissue sample, a biopsy specimen, a surgical specimen, a cytological specimen, whole blood, bone marrow, cerebral spinal fluid, peritoneal fluid, lymph fluid, serum, plasma, urine, stool, and nipple aspirate.
the at least 2 loci are selected from the group consisting of SEQ ID NO.s 1-485512. In some embodiments, the at least 2 loci are selected from the group consisting of the loci listed in TABLE 6. In some embodiments, the at least 2 loci are selected from the group consisting of the loci listed in TABLE 7. In some embodiments, the at least 2 loci are selected from the group consisting of the loci listed in TABLE 8. In some embodiments, the at least 2 loci are selected from the group consisting of the loci listed in TABLE 3.
the methylation states of at least 5 nucleic acid loci or at least 5 genes in said sample are determined. In some embodiments, the methylation states of at least 10 nucleic acid loci or at least 10 genes in said sample are determined. In some embodiments, the methylation states of at least 20 nucleic acid loci or at least 20 genes in said sample are determined. In some embodiments, the methylation states of at least 50 nucleic acid loci or at least 50 genes in said sample are determined. In some embodiments, the methylation states of at least 100 nucleic acid loci or at least 100 genes are determined.
the subject is a mammal. In some embodiments, the subject is a human.
nucleic acid loci are selected from the group consisting of the loci listed in TABLE 6. In some embodiments, the nucleic acid loci are selected from the group consisting of the loci listed in TABLE 7. In some embodiments, the nucleic acid loci are selected from the group consisting of the loci listed in TABLE 8.
Some embodiments of the methods, compositions and kits provided herein include a method for diagnosing rheumatoid arthritis or osteoarthritis, determining a rheumatoid arthritis or osteoarthritis prognosis, or determining or predicting a response to treatment for rheumatoid arthritis or osteoarthritis in a subject comprising determining the methylation states of a plurality of nucleic acid loci or the methylation states of a plurality of genes in a sample of said subject to obtain a methylation profile; and determining whether said methylation profile is indicative of rheumatoid arthritis, osteoarthritis, a rheumatoid arthritis or osteoarthritis prognosis, or a response to treatment for rheumatoid arthritis or osteoarthritis.
Some embodiments also include comparing the methylation state of the plurality of nucleic acid loci or the methylation state of the plurality of genes in the sample from said subject with the methylation state of the plurality of nucleic acid loci or the methylation state of the plurality of genes in normal tissue, tissue from a subject without said known rheumatoid arthritis or osteoarthritis prognosis, or tissue from a subject without said known response to treatment for rheumatoid arthritis or osteoarthritis.
an increase or decrease in the extent of methylation of plurality of nucleic acid loci or of the methylation state of the plurality of genes compared to the extent of methylation of the plurality of nucleic acid loci or the extent of methylation of the plurality of genes in normal tissue, tissue from a subject without said known rheumatoid arthritis or osteoarthritis prognosis, or tissue from a subject without said known response to treatment for rheumatoid arthritis or osteoarthritis is indicative of the presence or absence of rheumatoid arthritis, osteoarthritis, a rheumatoid arthritis or osteoarthritis prognosis, or response to treatment for rheumatoid arthritis or osteoarthritis for the subject.
the methylation state of said plurality of nucleic acid loci or said plurality of genes is determined in fibroblasts from said subject. In some embodiments, the methylation state of said plurality of nucleic acid loci or said plurality of genes is determined in synoviocytes from said subject. In some embodiments, the methylation state of said plurality of nucleic acid loci or said plurality of genes is determined in fibroblast-like synoviocytes from said subject. In some embodiments, the methylation state of said plurality of nucleic acid loci or said plurality of genes is determined in rheumatoid arthritis fibroblast-like synoviocytes from said subject. In some embodiments, the methylation state of said plurality of nucleic acid loci or said plurality of genes is determined in osteoarthritis fibroblast-like synoviocytes from said subject.
the methylation state of said plurality of nucleic acid loci or said plurality of genes is determined in macrophage from said subject.
the methylation state of said plurality of nucleic acid loci or said plurality of genes is determined in peripheral blood cells from said subject.
the peripheral blood cell is selected from the group consisting of white blood cell, neutrophil, eosinophil, basophil, lymphocyte, B cell, plasma cell, T cell, natural killer cell, monocyte, and dendritic cell.
the sample is selected from the group consisting of a tissue sample, a frozen tissue sample, a biopsy specimen, a surgical specimen, a cytological specimen, whole blood, bone marrow, cerebral spinal fluid, peritoneal fluid, lymph fluid, serum, plasma, urine, stool, and nipple aspirate.
the plurality of nucleic acid loci is selected from the group consisting of SEQ ID NO.s 1-485512. In some embodiments, the plurality of nucleic acid loci is selected from the group consisting of the loci listed in TABLE 6. In some embodiments, the plurality of nucleic acid loci is selected from the group consisting of the loci listed in TABLE 7. In some embodiments, the plurality of nucleic acid loci is selected from the group consisting of the loci listed in TABLE 8.
the plurality genes is selected from the group consisting of the loci listed in TABLE 3.
the methylation states of at least 5 nucleic acid loci or at least 5 genes in said sample are determined. In some embodiments, the methylation states of at least 10 nucleic acid loci or at least 10 genes in said sample are determined. In some embodiments, the methylation states of at least 20 nucleic acid loci or at least 20 genes in said sample are determined. In some embodiments, the methylation states of at least 50 nucleic acid loci or at least 50 genes in said sample are determined. In some embodiments, the methylation states of at least 100 nucleic acid loci or at least 100 genes are determined.
the subject is a mammal. In some embodiments, the subject is a human.
Some embodiments of the methods, compositions and kits provided herein include a method of identifying a methylation profile indicative of rheumatoid arthritis, osteoarthritis, a rheumatoid arthritis or osteoarthritis prognosis, or a response to treatment for rheumatoid arthritis or osteoarthritis comprising determining the methylation states of a plurality of nucleic acid loci or the methylation states of a plurality of genes in a sample from a test subject with rheumatoid arthritis, osteoarthritis, a particular rheumatoid arthritis or osteoarthritis prognosis, or a particular response to treatment for rheumatoid arthritis or osteoarthritis; determining the methylation states of said plurality of nucleic acid loci or said plurality of genes in a sample from a control subject without rheumatoid arthritis, without osteoarthritis, without said rheumatoid arthritis or osteoarth
Some embodiments also include storing data representing said loci or said genes which are hypermethylated or hypomethylated in said sample from said test subject relative to said sample from said control subject on a non-transitory computer readable medium.
Some embodiments also include comparing the methylation states of a plurality of nucleic acid loci or the methylation states of a plurality of genes in a sample from a test subject with rheumatoid arthritis, osteoarthritis, a particular rheumatoid arthritis or osteoarthritis prognosis, or a particular response to treatment for rheumatoid arthritis or osteoarthritis with the methylation states of said plurality of nucleic acid loci or the methylation states of said plurality of genes in a sample from a control subject without rheumatoid arthritis, without osteoarthritis, without said rheumatoid arthritis or osteoarthritis prognosis or without said response to treatment for rheumatoid arthritis or osteoarthritis.
the methylation state of said plurality of nucleic acid loci or said plurality of genes in a sample from said test subject or said control subject is determined in fibroblasts from said test subject or said control subject. In some embodiments, the methylation state of said plurality of nucleic acid loci or said plurality of genes in a sample from said test subject or said control subject is determined in synoviocytes from said test subject or said control subject. In some embodiments, the methylation state of said plurality of nucleic acid loci or said plurality of genes in a sample from said test subject or control subject is determined in fibroblast-like synoviocytes from said test subject or said control subject.
the methylation state of said plurality of nucleic acid loci or said plurality of genes in a sample from said test subject or control subject is determined in rheumatoid arthritis fibroblast-like synoviocytes from said test subject or control subject. In some embodiments, the methylation state of said plurality of nucleic acid loci or said plurality of genes in a sample from said test subject or control subject is determined in osteoarthritis fibroblast-like synoviocytes from said test subject or control subject.
the methylation state of said plurality of nucleic acid loci or said plurality of genes in a sample from said test subject or control subject is determined in macrophage from said test subject or control subject.
the methylation state of said plurality of nucleic acid loci or said plurality of genes in a sample from said test subject or control subject is determined in peripheral blood cells from said test subject or control subject.
the peripheral blood cell is selected from the group consisting of white blood cell, neutrophil, eosinophil, basophil, lymphocyte, B cell, plasma cell, T cell, natural killer cell, monocyte, and dendritic cell.
the sample is selected from the group consisting of a tissue sample, a frozen tissue sample, a biopsy specimen, a surgical specimen, a cytological specimen, whole blood, bone marrow, cerebral spinal fluid, peritoneal fluid, lymph fluid, serum, plasma, urine, stool, and nipple aspirate.
the methylation states of at least 5 nucleic acid loci or at least 5 genes are determined. In some embodiments, the methylation states of at least 10 nucleic acid loci or at least 10 genes are determined. In some embodiments, the methylation states of at least 20 nucleic acid loci or at least 20 genes are determined. In some embodiments, the methylation states of at least 50 nucleic acid loci or at least 50 genes are determined. In some embodiments, the methylation states of at least 100 nucleic acid loci or at least 100 genes are determined.
the subject is a mammal. In some embodiments, the subject is a human.
Some embodiments of the methods, compositions and kits provided herein include a method for diagnosing rheumatoid arthritis or osteoarthritis, determining a rheumatoid arthritis or osteoarthritis prognosis, or determining or predicting a response to treatment for rheumatoid arthritis or osteoarthritis in a subject comprising accessing first data representing the methylation status of nucleic acid loci or the methylation status of at least one gene which are differentially methylated in individuals with rheumatoid arthritis, osteoarthritis a rheumatoid arthritis or osteoarthritis prognosis, or a response to treatment for rheumatoid arthritis or osteoarthritis wherein said data is stored on a non-transitory computer readable medium; instructing a computer to compare said first data to second data representing the methylation status of said nucleic acid loci or said at least one gene in a sample taken from said subject, wherein said data representing the methyl
Some embodiments also include diagnosing rheumatoid arthritis or osteoarthritis, determining a rheumatoid arthritis or osteoarthritis prognosis, or determining or predicting a response to treatment for rheumatoid arthritis or osteoarthritis in said subject if said first data representing the methylation status of said nucleic acid loci or said at least one gene in a sample taken from said subject are significantly similar to said second data representing nucleic acid loci or genes which are differentially methylated in individuals with rheumatoid arthritis, osteoarthritis a rheumatoid arthritis or osteoarthritis prognosis, or a response to treatment for rheumatoid arthritis or osteoarthritis.
Some embodiments of the methods, compositions and kits provided herein include a method for diagnosing rheumatoid arthritis or osteoarthritis, determining a rheumatoid arthritis or osteoarthritis prognosis, or determining or predicting a response to treatment for rheumatoid arthritis or osteoarthritis in a subject comprising: determining the methylation states of at least 2 loci selected from the group consisting of SEQ ID NO.s 1-485512 in a sample obtained from the subject.
Some embodiments also include comparing the methylation states of the at least 2 loci in the sample with the methylation states of the loci in normal tissue, tissue from a subject with a known prognosis, or tissue from a subject with a known response to treatment.
an increase or decrease in the extent of methylation of at least 2 loci compared to the extent of methylation of the loci in normal tissue, tissue from a subject with a known prognosis, or tissue from a subject with a known response to treatment is indicative of the presence or absence of rheumatoid arthritis, prognosis, or response to treatment for the subject.
the methylation states of said at least 2 loci are determined in fibroblasts from said subject. In some embodiments, the methylation states of said at least 2 loci are determined in synoviocytes from said subject. In some embodiments, the methylation states of said at least 2 loci are determined in fibroblast-like synoviocytes from said subject. In some embodiments, the methylations state of said at least 2 loci are determined in rheumatoid arthritis fibroblast-like synoviocytes from said subject. In some embodiments, the methylation states of said at least 2 loci are determined in osteoarthritis fibroblast-like synoviocytes from said subject.
the methylation states of said at least 2 loci are determined in macrophage from said subject.
the methylation states of said at least 2 loci are determined in peripheral blood cells from said subject.
the peripheral blood cell is selected from the group consisting of white blood cell, neutrophil, eosinophil, basophil, lymphocyte, B cell, plasma cell, T cell, natural killer cell, monocyte, and dendritic cell.
the sample is selected from the group consisting of a tissue sample, a frozen tissue sample, a biopsy specimen, a surgical specimen, a cytological specimen, whole blood, bone marrow, cerebral spinal fluid, peritoneal fluid, lymph fluid, serum, plasma, urine, stool, and nipple aspirate.
the at least 2 loci are selected from the group consisting of the loci listed in TABLE 6. In some embodiments, the at least 2 loci are selected from the group consisting of the loci listed in TABLE 7. In some embodiments, the at least 2 loci are selected from the group consisting of the loci listed in TABLE 8. In some embodiments, the methylation states of at least 5 loci selected from the group consisting of the loci listed in TABLE 6, TABLE 7, and TABLE 8 are determined. In some embodiments, the methylation states of at least 10 loci selected from the group consisting of the loci listed in TABLE 6, TABLE 7, and TABLE 8 are determined.
the methylation states of at least 20 loci selected from the group consisting of the loci listed in TABLE 6, TABLE 7, and TABLE 8 are determined. In some embodiments, the methylation states of at least 50 loci selected from the group consisting of the loci listed in TABLE 6, TABLE 7, and TABLE 8 are determined. In some embodiments, the methylation states of at least 100 loci selected from the group consisting of the loci listed in TABLE 6, TABLE 7, and TABLE 8 are determined.
the subject is a mammal. In some embodiments, the subject is a human.
Some embodiments of the methods, compositions and kits provided herein include a method for identifying a therapeutic agent for treating rheumatoid arthritis or osteoarthritis comprising contacting a cell with a test agent; and determining the methylation states in the contacted cell of at least 2 loci which have differential extents of methylation in individuals with rheumatoid arthritis or osteoarthritis relative to individuals without rheumatoid arthritis or osteoarthritis.
Some embodiments also include comparing the methylation states of the at least 2 loci in the contacted cell with the methylation states of the loci in a cell which was not contacted with the test agent; and selecting a test agent that increases or decreases the extent of methylation of the at least 2 loci in the cell contacted with the test agent compared to the extent of methylation of the at least 2 loci in a cell which was not contacted with the test agent such that the extent of methylation of the at least 2 loci in the cell contacted with the test agent is a methylation states associated with the absence of rheumatoid arthritis or osteoarthritis or with a reduction in the symptoms associated with rheumatoid arthritis osteoarthritis.
the at least 2 loci are selected from the group consisting of SEQ ID NO.s 1-485512. In some embodiments, the at least 2 loci are selected from the group consisting of the loci listed in TABLE 6. In some embodiments, the at least 2 loci are selected from the group consisting of the loci listed in TABLE 7. In some embodiments, the at least 2 loci are selected from the group consisting of the loci listed in TABLE 8.
the methylation states of at least 5 loci selected from the group consisting of the loci listed in TABLE 6, TABLE 7, and TABLE 8 are determined. In some embodiments, the methylation states of at least 10 loci selected from the group consisting of the loci listed in TABLE 6, TABLE 7, and TABLE 8 are determined. In some embodiments, the methylation states of at least 20 loci selected from the group consisting of the loci listed in TABLE 6, TABLE 7, and TABLE 8 are determined. In some embodiments, the methylation states of at least 50 loci selected from the group consisting of the loci listed in TABLE 6, TABLE 7, and TABLE 8 are determined. In some embodiments, the methylation states of at least 100 loci selected from the group consisting of the loci listed in TABLE 6, TABLE 7, and TABLE 8 are determined.
the cell comprises a fibroblast. In some embodiments, the cell comprises a synoviocyte. In some embodiments, the cell comprises a fibroblast-like synoviocyte. In some embodiments, the cell comprises a rheumatoid arthritis fibroblast-like synoviocyte. In some embodiments, the cell comprises an osteoarthritis fibroblast-like synoviocyte.
the cell comprises a macrophage.
the cell comprises a peripheral blood cell.
the peripheral blood cell is selected from the group consisting of white blood cell, neutrophil, eosinophil, basophil, lymphocyte, B cell, plasma cell, T cell, natural killer cell, monocyte, and dendritic cell.
the cell is mammalian. In some embodiments, the cell is a human.
a sample comprises the cell, the sample selected from the group consisting of a tissue sample, a frozen tissue sample, a biopsy specimen, a surgical specimen, a cytological specimen, whole blood, bone marrow, cerebral spinal fluid, peritoneal fluid, lymph fluid, serum, plasma, urine, stool, and nipple aspirate.
kits for diagnosing rheumatoid arthritis or osteoarthritis include a kit for diagnosing rheumatoid arthritis or osteoarthritis, determining a rheumatoid arthritis or osteoarthritis prognosis, or determining or predicting a response to treatment for rheumatoid arthritis or osteoarthritis in a subject comprising: a reagent for determining the methylation states of at least 2 loci which have differential extents of methylation in individuals with rheumatoid arthritis or osteoarthritis relative to individuals without rheumatoid arthritis or osteoarthritis.
the at least 2 loci are selected from the group consisting of SEQ ID NO.s 1-485512. In some embodiments, the at least 2 loci are selected from the group consisting of the loci listed in TABLE 6, TABLE 7, and TABLE 8.
Some embodiments also include at least one polynucleotide primer comprising a sequence hybridizing to at least a portion of the at least 2 loci selected from the group consisting of the loci listed in TABLE 6, TABLE 7, and TABLE 8.
the reagent comprises a restriction enzyme.
the at least 2 loci are selected from the group consisting of the loci listed in TABLE 6. In some embodiments, the at least 2 loci are selected from the group consisting of the loci listed in TABLE 7. In some embodiments, the at least 2 loci are selected from the group consisting of the loci listed in TABLE 7. In some embodiments, the reagent can determine the methylation states of at least 5 loci selected from the group consisting of SEQ ID NO.s 1-485512. In some embodiments, the reagent can determine the methylation states of at least 5 loci selected from the group consisting of the loci listed in TABLE 6, TABLE 7, and TABLE 8.
the reagent can determine the methylation states of at least 10 loci selected from the group consisting of the loci listed in TABLE 6, TABLE 7, and TABLE 8. In some embodiments, the reagent can determine the methylation states of at least 20 loci selected from the group consisting of the loci listed in TABLE 6, TABLE 7, and TABLE 8. In some embodiments, the reagent can determine the methylation states of at least 50 loci selected from the group consisting of the loci listed in TABLE 6, TABLE 7, and TABLE 8. In some embodiments, the reagent can determine the methylation states of at least 100 loci selected from the group consisting of the loci listed in TABLE 6, TABLE 7, and TABLE 8.
Some embodiments of the methods, compositions and kits provided herein include a method for determining whether an individual suffers from rheumatoid arthritis or osteoarthritis comprising determining the methylation states of at least 2 loci which have differential extents of methylation in individuals with rheumatoid arthritis or osteoarthritis relative to individuals without rheumatoid arthritis or osteoarthritis, wherein the individual is diagnosed with rheumatoid arthritis if the methylation state of said at least 2 loci are associated with rheumatoid arthritis and the individual is diagnosed with osteoarthritis if the methylation states of said at least 2 loci are associated with osteoarthritis.
the at least 2 loci are selected from the group consisting of SEQ ID NO.s 1-485512. In some embodiments, the at least 2 loci are selected from the group consisting of the loci listed in TABLE 6. In some embodiments, the at least 2 loci are selected from the group consisting of the loci listed in TABLE 7. In some embodiments, the at least 2 loci are selected from the group consisting of the loci listed in TABLE 8.
the methylation states of at least 5 loci selected from the group consisting of the loci listed in TABLE 6, TABLE 7, and TABLE 8 are determined. In some embodiments, the methylation states of at least 10 loci selected from the group consisting of the loci listed in TABLE 6, TABLE 7, and TABLE 8 are determined. In some embodiments, the methylation states of at least 20 loci selected from the group consisting of the loci listed in TABLE 6, TABLE 7, and TABLE 8 are determined. In some embodiments, the methylation states of at least 50 loci selected from the group consisting of the loci listed in TABLE 6, TABLE 7, and TABLE 8 are determined. In some embodiments, the methylation states of at least 100 loci selected from the group consisting of the loci listed in TABLE 6, TABLE 7, and TABLE 8 are determined.
Some embodiments of the methods, compositions and kits provided herein include a method for diagnosing rheumatoid arthritis or osteoarthritis, determining a rheumatoid arthritis or osteoarthritis prognosis, or determining or predicting a response to treatment for rheumatoid arthritis or osteoarthritis in a subject comprising: determining the methylation states of at least 2 genes selected from the group consisting of the genes listed in TABLE 3 in a sample obtained from the subject.
Some embodiments also include comparing the methylation states of the at least 2 genes in the sample with the methylation states of the genes in normal tissue, tissue from a subject with a known prognosis, or tissue from a subject with a known response to treatment.
an increase or decrease in the extent of methylation of at least 2 genes compared to the extent of methylation of the of the genes in normal tissue, tissue from a subject with a known prognosis, or tissue from a subject with a known response to treatment is indicative of the presence or absence of rheumatoid arthritis, prognosis, or response to treatment for the subject.
the methylation states of said at least 2 loci are determined in fibroblasts from said subject. In some embodiments, the methylation states of said at least 2 loci are determined in synoviocytes from said subject. In some embodiments, the methylation states of said at least 2 loci are determined in fibroblast-like synoviocytes from said subject. In some embodiments, the methylation states of said at least 2 loci are determined in rheumatoid arthritis fibroblast-like synoviocytes from said subject. In some embodiments, the methylation states of said at least 2 loci are determined in osteoarthritis fibroblast-like synoviocytes from said subject.
the methylation states of said at least 2 loci are determined in macrophage from said subject.
the methylation states of said at least 2 loci are determined in peripheral blood cells from said subject.
the peripheral blood cell is selected from the group consisting of white blood cell, neutrophil, eosinophil, basophil, lymphocyte, B cell, plasma cell, T cell, natural killer cell, monocyte, and dendritic cell.
the sample is selected from the group consisting of a tissue sample, a frozen tissue sample, a biopsy specimen, a surgical specimen, a cytological specimen, whole blood, bone marrow, cerebral spinal fluid, peritoneal fluid, lymph fluid, serum, plasma, urine, stool, and nipple aspirate.
the methylation states of at least 5 genes selected from the group consisting of the genes listed in TABLE 3 are determined. In some embodiments, the methylation states of at least 10 genes selected from the group consisting of the genes listed in TABLE 3 are determined. In some embodiments, the methylation states of at least 20 genes selected from the group consisting of the genes listed in TABLE 3 are determined. In some embodiments, the methylation states of at least 50 genes selected from the group consisting of the genes listed in TABLE 3 are determined.
the subject is a mammal. In some embodiments, the subject is a human.
Some embodiments of the methods, compositions and kits provided herein include a method for identifying a therapeutic agent for treating rheumatoid arthritis or osteoarthritis comprising contacting a cell with a test agent; and determining the methylation states of at least 2 genes selected from the group consisting of the genes listed in TABLE 3 in the contacted cell.
Some embodiments also include comparing the methylation states of the at least 2 genes in the contacted cell with the methylation states of the genes in a cell which was not contacted with the test agent; and selecting a test agent that increases or decreases the extent of methylation of the at least 2 genes in the cell contacted with the test agent compared to the extent of methylation of the at least 2 genes in a cell which was not contacted with the test agent such that the extent of methylation of the at least 2 genes in the cell contacted with the test agent is a methylation states associated with the absence of rheumatoid arthritis or osteoarthritis or with a reduction in the symptoms associated with rheumatoid arthritis or osteoarthritis.
the methylation states of at least 5 genes selected from the group consisting of the genes listed in TABLE 3 are determined. In some embodiments, the methylation states of at least 10 genes selected from the group consisting of the genes listed in TABLE 3 are determined. In some embodiments, the methylation states of at least 20 genes selected from the group consisting of the genes listed in TABLE 3 are determined. In some embodiments, the methylation states of at least 50 genes selected from the group consisting of the genes listed in TABLE 3 are determined.
the cell comprises a fibroblast. In some embodiments, the cell comprises a synoviocyte. In some embodiments, the cell comprises a fibroblast-like synoviocyte. In some embodiments, the cell comprises a rheumatoid arthritis fibroblast-like synoviocyte. In some embodiments, the cell comprises an osteoarthritis fibroblast-like synoviocyte.
the cell comprises a macrophage.
the cell comprises a peripheral blood cell.
the peripheral blood cell is selected from the group consisting of white blood cell, neutrophil, eosinophil, basophil, lymphocyte, B cell, plasma cell, T cell, natural killer cell, monocyte, and dendritic cell.
the cell is mammalian. In some embodiments, the cell is a human.
a sample comprises the cell, the sample selected from the group consisting of a tissue sample, a frozen tissue sample, a biopsy specimen, a surgical specimen, a cytological specimen, whole blood, bone marrow, cerebral spinal fluid, peritoneal fluid, lymph fluid, serum, plasma, urine, stool, and nipple aspirate.
kits for diagnosing rheumatoid arthritis or osteoarthritis comprising: a reagent for determining the methylation states of at least 2 genes selected from the group consisting of the genes listed in TABLE 3.
Some embodiments also include at least one polynucleotide primer comprising a sequence hybridizing to at least a portion of the at least 2 genes selected from the group consisting of the genes listed in TABLE 3.
the reagent comprises a restriction enzyme.
the reagent can determine the methylation states of at least 5 genes selected from the group consisting of the genes listed in TABLE 3. In some embodiments, the reagent can determine the methylation states of at least 10 genes selected from the group consisting of the genes listed in TABLE 3. In some embodiments, the reagent can determine the methylation states of at least 20 genes selected from the group consisting of the genes listed in TABLE 3. In some embodiments, the reagent can determine the methylation states of at least 50 genes selected from the group consisting of the genes listed in TABLE 3.
Some embodiments of the methods, compositions and kits provided herein include a method for identifying a therapeutic agent for treating rheumatoid arthritis or osteoarthritis comprising contacting a cell with a test agent; and determining the methylation states of at least 2 genes selected from a gene encoding a protein that acts in a pathway that includes a protein encoded by a gene that is differentially methylated in a rheumatoid arthritis cell or that is differentially methylated in a osteoarthritis cell compared to a normal cell.
Some embodiments also include comparing the methylation states of the at least 2 genes in the contacted cell with the methylation states of the genes in a cell which was not contacted with the test agent; and selecting a test agent that increases or decreases the extent of methylation of the at least 2 genes in the cell contacted with the test agent compared to the extent of methylation of the at least 2 genes in a cell which was not contacted with the test agent such that the extent of methylation of the at least 2 genes in the cell contacted with the test agent are methylation states associated with the absence of rheumatoid arthritis or methylation states associated with the absence of osteoarthritis with a reduction in the symptoms associated with rheumatoid arthritis.
the pathway is selected from the group consisting of the pathways listed in TABLE 5.
the cell comprises a fibroblast. In some embodiments, the cell comprises a synoviocyte. In some embodiments, the cell comprises a fibroblast-like synoviocyte. In some embodiments, the cell comprises a rheumatoid arthritis fibroblast-like synoviocyte. In some embodiments, the cell comprises an osteoarthritis fibroblast-like synoviocyte.
the cell comprises a macrophage.
the cell comprises a peripheral blood cell.
the peripheral blood cell is selected from the group consisting of white blood cell, neutrophil, eosinophil, basophil, lymphocyte, B cell, plasma cell, T cell, natural killer cell, monocyte, and dendritic cell.
the cell is mammalian. In some embodiments, the cell is a human.
a sample comprises the cell, the sample selected from the group consisting of a tissue sample, a frozen tissue sample, a biopsy specimen, a surgical specimen, a cytological specimen, whole blood, bone marrow, cerebral spinal fluid, peritoneal fluid, lymph fluid, serum, plasma, urine, stool, and nipple aspirate.
Some embodiments of the methods, compositions and kits provided herein include a method for identifying therapeutic agents for treating rheumatoid arthritis or osteoarthritis comprising contacting a cell with a test agent; and determining the activity of a protein encoded by a gene differentially methylated in a rheumatoid arthritis cell or that is differentially methylated in an osteoarthritis cell compared to a normal cell.
Some embodiments also include comparing the activity of the protein in the contacted cell with the activity of the protein in a cell which was not contacted with the test agent; and selecting a test agent that increases or decreases the activity of protein in the cell contacted with the test agent compared to the activity of the protein in a cell which was not contacted with the test agent such that the activity of the protein in the cell contacted with the test agent is an activity associated with the absence of rheumatoid arthritis or an activity associated with the absence of osteoarthritis with a reduction in the symptoms associated with rheumatoid arthritis.
the protein is encoded by a gene selected from the group consisting of the genes listed in TABLE 3.
the cell comprises a fibroblast. In some embodiments, the cell comprises a synoviocyte. In some embodiments, the cell comprises a fibroblast-like synoviocyte. In some embodiments, the cell comprises a rheumatoid arthritis fibroblast-like synoviocyte. In some embodiments, the cell comprises an osteoarthritis fibroblast-like synoviocyte.
the cell comprises a macrophage.
the cell comprises a peripheral blood cell.
the peripheral blood cell is selected from the group consisting of white blood cell, including neutrophil, eosinophil, basophil, lymphocyte, B cell, plasma cell, T cell, natural killer cell, monocyte, and dendritic cell.
the cell is mammalian. In some embodiments, the cell is a human.
Some embodiments of the methods, compositions and kits provided herein include a method of determining the methylation status of a plurality of human nucleic acid loci comprising contacting a nucleic acid sample from a human subject with a reagent capable of providing an indication of the methylation status of said loci, wherein said loci comprise at least 5 which have differential extents of methylation in individuals with rheumatoid arthritis or osteoarthritis relative to individuals without rheumatoid arthritis or osteoarthritis.
said reagent is a restriction enzyme.
said reagent is a primer.
said reagent is a probe.
said reagent comprises sodium bisulfate.
the at least 5 loci are selected from the group consisting of SEQ ID NO.s 1-485512. In some embodiments, the at least 5 loci are selected from the group consisting of the loci listed in TABLE 6, TABLE 7, and TABLE 8.
nucleic acid array consisting essentially of nucleic acids useful for diagnosing rheumatoid arthritis or osteoarthritis, determining a prognosis of rheumatoid arthritis or osteoarthritis, or determining or predicting a response to treatment of a subject being evaluated for or suffering from rheumatoid arthritis or osteoarthritis, wherein said nucleic acids comprise at least 5 loci which have differential extents of methylation in individuals with rheumatoid arthritis or osteoarthritis relative to individuals without rheumatoid arthritis or osteoarthritis.
said nucleic acids comprise at least 5 loci selected from the group consisting of SEQ ID NO.s 1-485512. In some embodiments, said nucleic acids comprise at least 5 loci selected from the group consisting of the loci listed in TABLE 6, TABLE 7, and TABLE 8. In some embodiments, said nucleic acids comprise at least 10 loci selected from the group consisting of the loci listed in TABLE 6, TABLE 7, and TABLE 8. In some embodiments, said nucleic acids comprise at least 20 loci selected from the group consisting of the loci listed in TABLE 6, TABLE 7, and TABLE 8. In some embodiments, said nucleic acids comprise at least 50 loci selected from the group consisting of the loci listed in TABLE 6, TABLE 7, and TABLE 8.
Some embodiments of the methods, compositions and kits provided herein include a method of ameliorating rheumatoid arthritis or osteoarthritis comprising evaluating the methylation status of a plurality of human nucleic acid loci in a nucleic acid sample from a human subject having symptoms of rheumatoid arthritis or osteoarthritis, wherein said loci comprise at least 5 loci which have differential extents of methylation in individuals with rheumatoid arthritis or osteoarthritis relative to individuals without rheumatoid arthritis or osteoarthritis; and administering a treatment for rheumatoid arthritis or osteoarthritis if said at least 5 loci have a methylation status indicative of rheumatoid arthritis or osteoarthritis.
said nucleic acids comprise at least 5 loci selected from the group consisting of SEQ ID NO.s 1-485512. In some embodiments, said nucleic acids comprise at least 5 loci selected from the group consisting of the loci listed in TABLE 6, TABLE 7, and TABLE 8. In some embodiments, said nucleic acids comprise at least 10 loci selected from the group consisting of the loci listed in TABLE 6, TABLE 7, and TABLE 8. In some embodiments, said nucleic acids comprise at least 20 loci selected from the group consisting of the loci listed in TABLE 6, TABLE 7, and TABLE 8. In some embodiments, said nucleic acids comprise at least 50 loci selected from the group consisting of the loci listed in TABLE 6, TABLE 7, and TABLE 8.
Some embodiments of the methods, compositions and kits provided herein include a mixture comprising a plurality of human nucleic acid loci from a human subject having symptoms indicative of potential rheumatoid arthritis or osteoarthritis and a reagent capable of providing an indication of the methylation status of said loci, wherein said loci comprise at least 5 loci which have differential extents of methylation in individuals with rheumatoid arthritis or osteoarthritis relative to individuals without rheumatoid arthritis or osteoarthritis.
said nucleic acids comprise at least 5 loci selected from the group consisting of SEQ ID NO.s 1-485512. In some embodiments, said nucleic acids comprise at least 5 loci selected from the group consisting of the loci listed in TABLE 6, TABLE 7, and TABLE 8. In some embodiments, said nucleic acids comprise at least 10 loci selected from the group consisting of the loci listed in TABLE 6, TABLE 7, and TABLE 8. In some embodiments, said nucleic acids comprise at least 20 loci selected from the group consisting of the loci listed in TABLE 6, TABLE 7, and TABLE 8. In some embodiments, said nucleic acids comprise at least 50 loci selected from the group consisting of the loci listed in TABLE 6, TABLE 7, and TABLE 8
FIG. 1 depicts DNA methyltranserases (DNMTs) function.
DNMTs transfer methyl groups from SAM to deoxycytodine.
DNMT function can be affected by changes in methyl donors in the diet or by DNMT inhibitors, like 5-aza-2′-deoxycytidine (5-azaC).
5-aza-2′-deoxycytidine 5-aza-2′-deoxycytidine
the analog 5-azacytidine is also incorporated into RNA and can interfere with many other cellular processes.
FIG. 2 depicts an ILLUMINA array analysis of DNA methylation in RA and OA FLS, and the hierarchical clustering and heatmap of differentially methylated loci.
the methylation levels at the 1,859 significantly differentially methylated loci were used for hierarchical clustering.
the clustering of the sample is shown by the dendrogram at the top and the clustering of the loci is shown by the dendrogram on the left.
the methylation levels at the loci are shown in the heatmap.
FIG. 3 depicts hypomethylation of several genes in RA FLS basal expression compared to OA FLS.
Gene expression was determined by PCR in 6 to 13 separate OA and RA FLS lines for 7 genes that were significantly hypomethylated (CHI3L1, COL1A1, MYEF2, ITG4A, SYNJ2, STK24, MAP3K5).
CHI3L1, COL1A1, MYEF2, ITG4A, SYNJ2, STK24, MAP3K5 As a group, expression of hypomethylated genes in RA was significantly greater than OA (P ⁇ 0.01); expression of genes that were normally methylated in RA was similar to OA, e.g., AXIN, IKKE, TBK1, NANOG, POU5F1, MAP2K6, IRF3.
FIG. 5 depicts a Cytoscape analysis to evaluate the networks affected by differential methylation and determine the most likely targets for subsequent analysis.
FIG. 6 depicts basal expression of DNMTs. DNMT expression was examined in resting cultured FLS. Synoviocytes were isolated from RA and OA synovium at the time of total joint replacement. 4 th through 6 th passage cells were evaluated by qPCR (graph shows relative expression units using the standard curve method).
FIG. 7 depicts decreased DNMT1 gene expression after IL-1 stimulation. FLS were stimulated with IL-1 for 24 hr and DNMT expression was determined by qPCR.
FIG. 8 depicts the decrease in DNMT function in FLS in response to IL-1.
FLS were stimulated with 1 ng/ml of IL-1 for 14 days and extracts were assayed using the DNMT Activity/Inhibition Assay (Active Motif Co., Carlsbad, Calif.), which is an ELISA-based method that measures methylation of a CpG-enriched DNA substrate.
DNMT Activity/Inhibition Assay Active Motif Co., Carlsbad, Calif.
Peripheral blood mononuclear cells in patients with rheumatoid arthritis demonstrate global methylation abnormalities that parallel those found in cultured fibroblast-like synoviocytes, confirming that peripheral blood cells reflect synovial biology and synoviocyte function.
Methylation of the IL-10 promoter has been studied in patients with RA (Fu L. H. et al., Methylation status of the IL-10 gene promoter in the peripheral blood mononuclear cells of rheumatoid arthritis patients. Yi Chuan. 2007 November; 29(11):1357-61).
the unique pattern of DNA methylation in RA or OA has several implications.
the pattern can be used for several applications, including: diagnosis of RA or OA; assessment of disease activity and prognosis of RA or OA; identification of novel therapeutic targets useful for the development of novel therapies for RA or OA; and the development of novel therapies that increase or decrease DNA methylation and alter the pattern, such as through DNMT inhibitors or activators.
Embodiments of the present invention include methods, compositions and kits for evaluating a diagnosis, prognosis, or response to treatment of a subject with a disorder such as rheumatoid arthritis or osteoarthritis. Some embodiments include identifying a therapeutic agent for treating a disorder such as rheumatoid arthritis or osteoarthritis.
rheumatoid FLS Like peripheral blood mononuclear cells, rheumatoid FLS exhibit an abnormal phenotype that contributes to disease pathogenesis (Firestein, G S. Invasive fibroblast-like synoviocytes in rheumatoid arthritis: Passive responders or transformed aggressors? Arthritis Rheum 39:1781-1790, 1996). Functional studies suggest that RA FLS are imprinted in situ and maintain these features after many passages in tissue culture. For example, RA FLS, unlike OA or normal synoviocytes, adhere to and invade cartilage explants in SCID mice (Müller-Ladner U et al.
rheumatoid arthritis Several mechanisms have been implicated in the rheumatoid phenotype. For instance, resistance to apoptosis can be due, in part, to defective expression of Phosphatase and tensin homolog (PTEN) expression, high levels of sentrin, or preferential shunting of stressed cells to DNA repair rather than programmed cell death (Pap T et al. Activation of synovial fibroblasts in rheumatoid arthritis: lack of Expression of the tumour suppressor PTEN at sites of invasive growth and destruction. Arthritis Res. 2000; 2(1):59-64; Franz J K et al.
PTEN Phosphatase and tensin homolog
the abnormal cells are more invasive and produce increased amounts of cytokines and metalloproteinases.
Microdissection of rheumatoid synovium shows islands of mutant cells residing in the intimal lining that produce prodigious amount of IL-6 (Yamanishi Y et al. p53 regulates apoptosis, synovitis and joint destruction in collagen-induced arthritis. Amer J Pathol, 160:123-30, 2002).
Microsatellite instability has also been identified in RA synovium, in part due to decreased DNA repair function (Lee S—H et al. Microsatellite instability and suppressed DNA repair enzyme expression in rheumatoid arthritis. J Immunol, 170:2214-20, 2003).
HATs histone acetyltransferases
HDACs histone deacetylases
MicroRNAs are another epigenetic mechanism that contribute to DNA accessibility and chromatin remodeling by directly targeting individual genes. Expression of some individual microRNAs like microRNA-124a, are decreased in RA compared with OA cells, leading to enhanced chemokine expression (Nakamachi Y et al.
MicroRNA-124a is a key regulator of proliferation and monocyte chemoattractant protein 1 secretion in fibroblast-like synoviocytes from patients with rheumatoid arthritis. Arthritis Rheum 60:1294, 2009; Stanczyk J et al. Altered expression of microRNA-203 in rheumatoid arthritis synovial fibroblasts and its role in fibroblast activation. Arthritis Rheum. 2011 February; 63 (2): 373-81).
DNA methylation is especially relevant to RA in terms of epigenetic mechanisms by virtue of its role in neoplasia as well as embryonic growth and development.
Normal ontogeny relies on a carefully orchestrated sequence of DNA methylation to repress regulatory genes by methylating cytosine in CpG islands after they have completed their programmed role in early development (Christophersen N S and Helin K. Epigenetic control of embryonic stem cell fate. J Exp Med. Oct. 25, 2010; 207(11):2287-95).
Methylation abnormalities have been associated with a variety of diseases, most notably cancer where hypomethylation and renewed expression of embryonic genes can allow cells to de-differentiate and escape from normal homeostatic controls (Kulis M and Esteller M. DNA methylation and cancer. Adv Genet. 2010; 70:27-56).
Hypermethylation has also been associated with certain malignancies (Ren J et al. DNA hypermethylation as a chemotherapy target. Cell Signal. Feb. 21, 2011).
DNMTs DNA methyltransferases
FIG. 1 DNA methyltransferases
DNMT1, DNMT3a, and DNMT3b are the primary enzymes responsible for CpG methylation.
DNMT3a and DNMT3b mainly regulate de novo methylation while DNMT1 maintains methylation, especially during cell division.
DNMT1 plays a greater role perpetuating methylation patterns in proliferating cells.
Decreased DNMT expression is associated with global hypomethylation as well as suppressed methylation of individual genes that participate in malignant transformation (Shukla V et al. BRCA1 affects global DNA methylation through regulation of DNMT1. Cell Res. 2010 November; 20(11):1201-15).
DNMT expression and DNA methylation is not fixed but can be influenced by the environment and modify gene expression throughout life. The DNMTs can also maintain the methylation pattern during cell division, thereby allowing daughter cells to reflect the environmental influences of the parent cells.
DNMTs can also permit vertical transmission of parental DNA methylation (Ko Y G et al. Stage-by-stage change in DNA methylation status of Dnmt1 locus during mouse early development. J Biol. Chem. 2005 Mar. 11; 280(10):9627-34). This process allows relatively rapid responses to environmental stress that can persist over many cell divisions and even across generations (Rosenfeld. Animal models to study environmental epigenetics. Biol Reprod. 2010, 82:473-88; Kaati G et al. Transgenerational response to nutrition, early life circumstances and longevity. Eur J Hum Genet. 2007 July; 15(7):784-90).
T cells can be affected by DNA methylation and influence Th2 differentiation (Gamper C J et al. Identification of DNA methyltransferase 3a as a T cell receptor-induced regulator of Th1 and Th2 differentiation. J Immunol. 2009 Aug. 15; 183(4):2267-76).
the DNMT inhibitor 5′-aza-2′-deoxycytidine (5-azaC) (Fandy T E. Development of DNA methyltransferase inhibitors for the treatment of neoplastic diseases. Curr Med. Chem.
T cell genes including IFN ⁇ , IL-4, CD70, and LFA-1.
5-azaC enhances autoreactivity and induces robust responses to normally sub-threshold stimulation (Richardson B. DNA methylation and autoimmune disease. Clin Immunol. 2003 October; 109(1):72-9).
T and B cell interactions are also affected, in part due to altered expression of surface receptors like CD70 (Oelke K et al. Overexpression of CD70 and overstimulation of IgG synthesis by lupus T cells and T cells treated with DNA methylation inhibitors. Arthritis Rheum. 2004 June; 50(6):1850-60).
DNMT regulation and the methylation status of FLS were examined in view of the association of DNA hypomethylation and an aggressive phenotype in cancer. As shown in this application, it was found that IL-1 significantly decreased DNMT1, DNMT3a, and DNMT3b gene expression within hours. The change in DNMT expression was accompanied by decreased DNMT function in nuclear extracts and global hypomethylation. More striking, an ILLUMINA methylation array study of RA and OA cells showed 100% concordance between DNA methylation patterns and the presence of RA.
the unique patterns of DNA methylation in RA or OA have several implications.
the patterns can be used for several applications, including: diagnosis of RA or OA; assessment of disease activity and prognosis of RA or OA; identification of novel therapeutic targets useful for the development of novel therapies for RA or OA; and the development of novel therapies that increase or decrease DNA methylation and alter the pattern, such as though DNMT inhibitors or activators.
methylation refers to cytosine methylation at positions C5 or N4 of cytosine, the N6 position of adenine or other types of nucleic acid methylation.
“methylation” refers to cytosine methylation at positions C5 of cytosine, namely, 5-methly cytosine.
In vitro amplified DNA is unmethylated because in vitro DNA amplification methods do not retain the methylation pattern of the amplification template.
unmethylated DNA or “methylated DNA” can also refer to amplified DNA whose original template was unmethylated or methylated, respectively.
a “methylation profile” refers to a set of data representing the methylation states of two or more loci within a molecule of DNA from e.g., the genome of an individual or cells or tissues from an individual.
the profile can indicate the methylation state of every cytosine base in an individual, can comprise information regarding a subset of the base pairs (e.g., the methylation state of specific restriction enzyme recognition sequence) in a genome, or can comprise information regarding regional methylation density of each locus.
methylation status refers to the presence, absence and/or quantity of methylation at a particular nucleotide, or nucleotides within a portion of DNA. Determination of the methylation status of a particular DNA sequence (e.g., a locus, a DNA biomarker or DNA region as described herein) can involve determination of the methylation state of every cytosine in the sequence or can involve determination of the methylation state of a subset of the cytosines (such as the methylation state of cytosines in one or more specific restriction enzyme recognition sequences) within the sequence, or can involve determining regional methylation density within the sequence without providing precise information of where in the sequence the methylation occurs.
a particular DNA sequence e.g., a locus, a DNA biomarker or DNA region as described herein
Determination of the methylation status of a particular DNA sequence can involve determination of the methylation state of every cytosine in the sequence or can involve determination of the methylation state of
the methylation status can optionally be represented or indicated by a “methylation value.”
a methylation value can be generated, for example, by quantifying the amount of intact DNA present following restriction digestion with a methylation dependent restriction enzyme.
a value i.e., a methylation value, for example from the above described example, represents the methylation status and can thus be used as a quantitative indicator of methylation status. This is of particular use when it is desirable to compare the methylation status of a sequence in a sample to a threshold value.
methylation-dependent restriction enzyme refers to a restriction enzyme that cleaves or digests DNA at or in proximity to a methylated recognition sequence, but does not cleave DNA at or near the same sequence when the recognition sequence is not methylated.
Methylation-dependent restriction enzymes include those that cut at a methylated recognition sequence (e.g., DpnI) and enzymes that cut at a sequence near but not at the recognition sequence (e.g., McrBC).
McrBC's recognition sequence is 5′ RmC(N40-3000) RmC 3′ where “R” is a purine and “mC” is a methylated cytosine and “N40-3000” indicates the distance between the two RmC half sites for which a restriction event has been observed.
McrBC generally cuts close to one half-site or the other, but cleavage positions are typically distributed over several base pairs, approximately 30 base pairs from the methylated base. McrBC sometimes cuts 3′ of both half sites, sometimes 5′ of both half sites, and sometimes between the two sites.
Exemplary methylation-dependent restriction enzymes include, e.g., McrBC (see, e.g., U.S. Pat. No.
methylation-sensitive restriction enzyme refers to a restriction enzyme that cleaves DNA at or in proximity to an unmethylated recognition sequence but does not cleave at or in proximity to the same sequence when the recognition sequence is methylated.
Exemplary methylation-sensitive restriction enzymes are described in, e.g., McClelland et al., Nucleic Acids Res. 22(17):3640-59 (1994) and http://rebase.neb.com.
Suitable methylation-sensitive restriction enzymes that do not cleave DNA at or near their recognition sequence when a cytosine within the recognition sequence is methylated at position C5 include, e.g., Aat I I, Aci I, Acd I, Age I, Alu I, Asc I, Ase I, AsiS I, Bbe I, BsaA I, BsaH I, BsiE I, BsiW I, BsrF I, BssH II, BssK I, BstB I, BstN I, BstU I, Cla I, Eae I, Eag I, Fau I, Fse I, Hha I, HinP1 I, HinC II, Hpa II, Hpy99 I, HpyCH4 IV, Kas I, Mbo I, Mlu I, MapA1 I, Msp I, Nae I, Nar I, Not I, Pml I, Pst I, Pvu I, Rsr II, Sac
Suitable methylation-sensitive restriction enzymes that do not cleave DNA at or near their recognition sequence when an adenosine within the recognition sequence is methylated at position N6 include, e.g., Mbo I.
any methylation-sensitive restriction enzyme including homologs and orthologs of the restriction enzymes described herein, is also suitable for use in the present invention.
a methylation-sensitive restriction enzyme that fails to cut in the presence of methylation of a cytosine at or near its recognition sequence may be insensitive to the presence of methylation of an adenosine at or near its recognition sequence.
a methylation-sensitive restriction enzyme that fails to cut in the presence of methylation of an adenosine at or near its recognition sequence may be insensitive to the presence of methylation of a cytosine at or near its recognition sequence.
Sau3AI is sensitive (i.e., fails to cut) to the presence of a methylated cytosine at or near its recognition sequence, but is insensitive (i.e., cuts) to the presence of a methylated adenosine at or near its recognition sequence.
methylation-sensitive restriction enzymes are blocked by methylation of bases on one or both strands of DNA encompassing of their recognition sequence, while other methylation-sensitive restriction enzymes are blocked only by methylation on both strands, but can cut if a recognition site is hemi-methylated.
diagnosing can include determining whether a methylation status of 1 or more loci is indicative of a disorder, such as rheumatoid arthritis.
determining a prognosis can include determining whether methylation status of 1 or more loci is indicative of a likelihood of improvement in symptoms of a disorder, such as rheumatoid arthritis.
determining or predicting a response to treatment can include determining whether methylation status of 1 or more loci after treatment is more similar to a normal status before treatment or earlier in the treatment regimen.
the 1 or more locus or 1 or more gene is a locus or gene with no known association with RA.
the methylation state of at least 1 locus or at least one gene selected from a locus or gene described herein in a sample obtained from a subject is determined.
loci for which the methylation state may be evaluated include the loci listed in TABLE 6, TABLE 7, and TABLE 8.
loci for which the methylation state may be evaluated include SEQ ID NO.s:1-485512. In each sequence provided in SEQ ID NO.s 1-485512, the “C” which is potentially methylated is at position 61.
genes for which the methylation state may be evaluated include the loci listed in TABLE 3.
additional loci and genes useful for the methods and compositions provided herein can be further identified using the methods described herein.
additional loci and genes useful for the methods and compositions provided herein are identified by conducting methylation analyses in additional samples, thereby providing an increased number of data points which could assist in the identification of further genes or loci having statistically significant differences in their methylation states.
the sample can comprise an in vivo sample, an in vitro sample, or an ex vivo sample. It will be understood, that in some embodiments of the compositions or methods provided herein, a sample or cell can be in vivo.
a sample or cell can be ex vivo.
Methods to determine the methylation state of at least one locus or at least one gene are well known in the art and examples are provided herein.
the subject is a mammal, such as a human.
the methylation states of at least about 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 loci or more than 500 loci are determined.
Some embodiments also include comparing the methylation state of the at least 1 locus in the sample with the methylation state of the locus in a normal cell, cell from a subject with a known prognosis, or cell from a subject with a known response to treatment.
the methylation states of at least about 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 genes or more than 500 genes are determined.
Some embodiments also include comparing the methylation state of the at least 1 gene in the sample with the methylation state of the gene in a normal cell, cell from a subject with a known prognosis, or cell from a subject with a known response to treatment.
an increase or decrease in the methylation state of the at least 1 locus compared to the methylation state of the locus in normal cell, cell from a subject with a known prognosis, or cell from a subject with a known response to treatment is indicative of the diagnosis, prognosis, or response to treatment for the subject.
an increase or decrease in the methylation state of a locus selected from the group consisting of the loci listed in TABLE 6 is indicative of the diagnosis, prognosis, or response to treatment for the subject.
an increase or decrease in the methylation state of a locus selected from the group consisting of the loci listed in TABLE 7 is indicative of the diagnosis, prognosis, or response to treatment for the subject.
an increase or decrease in the methylation state of a locus selected from the group consisting of the loci listed in TABLE 8 is indicative of the diagnosis, prognosis, or response to treatment for the subject.
an increase or decrease in the methylation state of the at least 1 gene compared to the methylation state of the gene in a normal cell, cell from a subject with a known prognosis, or tissue from a subject with a known response to treatment is indicative of the diagnosis, prognosis, or response to treatment for the subject.
an increase or decrease in the methylation state of a gene selected from the group consisting of the genes listed in TABLE 3 is indicative of the diagnosis, prognosis, or response to treatment for the subject.
the increase or decrease in methylation occurs in a cell, such as a synoviocyte, such as a fibroblast-like synoviocyte, for example, a rheumatoid arthritis fibroblast-like synoviocyte or an osteoarthritis fibroblast-like synoviocyte.
the cell comprises macrophage.
the cell comprises a peripheral blood cell.
‘peripheral blood cell’ can include a cellular component of blood which contains DNA. Examples of peripheral blood cells include white blood cells, including neutrophils, eosinophils, basophils, lymphocytes, B cell, plasma cells, T cells, natural killer cells, monocytes, and dendritic cells.
the cell is mammalian, e.g., human.
the loci and genes which are differentially methylated in fibroblast-like synoviocytes from individuals with rheumatoid arthritis or osteoarthritis and the loci and genes which are differentially methylated in individuals with rheumatoid or osteoarthritis in cell types other than fibroblast-like synoviocytes may partially overlap. However, it is likely that there will be loci and genes which exhibit differential methylation in individuals with rheumatoid arthritis or osteoarthritis in one cell type which are not differentially methylated in other cell types from individuals with rheumatoid arthritis or osteoarthritis.
differentially methylated loci and genes identified in T cells and in B cells types from individuals with rheumatoid arthritis or osteoarthritis can include loci and genes that are different in each cell type. Differentially methylated loci and genes in different cell types from individuals with rheumatoid arthritis or osteoarthritis can be identified using the methods described herein.
Some embodiments include methods of ameliorating rheumatoid arthritis or osteoarthritis in a subject. Some such embodiments include evaluating the methylation status of a plurality of human nucleic acid loci in a nucleic acid sample from a human subject having symptoms of rheumatoid arthritis or osteoarthritis.
the loci comprise at least about 5 loci, at least about 10 loci, at least about 15 loci, at least about 20 loci, at least about 25 loci, at least about 50 loci, and at least about 100 loci.
the loci are selected from the group consisting of the loci listed in TABLE 6, TABLE 7, or TABLE 8.
the loci may be selected from SEQ ID NO.s:1-485512. Some embodiments also include administering a treatment for rheumatoid arthritis or osteoarthritis if the loci have a methylation status indicative of rheumatoid arthritis or osteoarthritis.
Some embodiments include a mixture comprising a plurality of human nucleic acid loci from a human subject having symptoms indicative of potential rheumatoid arthritis or osteoarthritis and a reagent capable of providing an indication of the methylation status of said loci.
the loci comprise at least about 5 loci, at least about 10 loci, at least about 15 loci, at least about 20 loci, at least about 25 loci, at least about 50 loci, and at least about 100 loci.
the loci are selected from the group consisting of the loci listed in TABLE 6, TABLE 7, or TABLE 8.
the loci are selected from SEQ ID NO.s:1-485512
the methylation state of more than one DNA region e.g., gene, locus or portion thereof is determined.
the methylation state of a DNA region or portion thereof is determined and then normalized (e.g., compared) to the methylation state of a control locus.
the control locus will have a known, relatively constant, methylation status.
the control sequence can be previously determined to have no, some or a high amount of methylation, thereby providing a relative constant value to control for error in detection methods, etc., unrelated to the presence or absence of a disorder.
the control locus is endogenous, i.e., is part of the genome of the individual sampled.
testes-specific histone 2B gene (hTH2B in human) gene is known to be methylated in all somatic tissues except testes.
control locus can be an exogenous locus, i.e., a DNA sequence spiked into the sample in a known quantity and having a known methylation status.
a DNA region comprises a nucleic acid including one or more methylation sites of interest (e.g., a cytosine, a “microarray feature,” or an amplicon amplified from select primers) and flanking nucleic acid sequences (i.e., “wingspan”) of up to 4 kilobases (kb) in either or both of the 3′ or 5′ direction from the amplicon.
methylation sites of interest e.g., a cytosine, a “microarray feature,” or an amplicon amplified from select primers
flanking nucleic acid sequences i.e., “wingspan”
kb kilobases
the wingspan of the one or more DNA regions is about 0.5 kb, 0.75 kb, 1.0 kb, 1.5 kb, 2.0 kb, 2.5 kb, 3.0 kb, 3.5 kb or 4.0 kb in both 3′ and 5′ directions relative to the sequence represented by the microarray feature.
the DNA region of interest can comprise and/or be immediately adjacent to a locus selected from the group consisting of the loci listed in TABLE 6, TABLE 7, or TABLE 8, or a gene selected from a gene listed in TABLE 3.
the locus may be selected from SEQ ID NO.s:1-485512.
the nucleic acid sequences of the loci listed in TABLE 6, TABLE 7, and TABLE 8 are available, for example, in the Illumina CpG database, and included in SEQ ID NO.s:1-485512.
the methylation sites in a DNA region can reside in non-coding transcriptional control sequences (e.g., promoters, enhancers, etc.) or in coding sequences, including introns and exons of the loci listed in TABLE 6, TABLE 7, or TABLE 8, and genes listed in TABLE 3.
the methods comprise detecting the methylation status in the promoter regions (e.g., comprising the nucleic acid sequence that is about 1.0 kb, 1.5 kb, 2.0 kb, 2.5 kb, 3.0 kb, 3.5 kb or 4.0 kb 5′ from the transcriptional start site through to the translational start site) of one or more of the locus identified in TABLE 6, TABLE 7, TABLE 8, or TABLE 3.
the locus may be selected from SEQ ID NO.s:1-485512.
an array can be used to determine the methylation state of at least one locus, such as the ILLUMINA HumanMethylation 450 BeadChip.
DNA is treated with bisulfite to convert unmethylated cytosines to uracil, methylated cytosines are protected and remain cytosine.
a determination step is performed to identify whether a base at a particular locus was converted. Methylation status of the interrogated site is calculated as the ratio of the signal from a methylated probe relative to the sum of both methylated and unmethlylated probes.
Arrays such as the ILLUMINA HumanMethylation 450 BeadChip, include genes and CpG islands and other sequences.
methods for detecting methylation include randomly shearing or randomly fragmenting the genomic DNA, cutting the DNA with a methylation-dependent or methylation-sensitive restriction enzyme and subsequently selectively identifying and/or analyzing the cut or uncut DNA.
Selective identification can include, for example, separating cut and uncut DNA (e.g., by size) and quantifying a sequence of interest that was cut or, alternatively, that was not cut. See, e.g., U.S. Pat. No. 7,186,512.
the method can encompass amplifying intact DNA after restriction enzyme digestion, thereby only amplifying DNA that was not cleaved by the restriction enzyme in the area amplified. See, e.g., U.S. patent application Ser. Nos.
amplification can be performed using primers that are gene specific.
adaptors can be added to the ends of the randomly fragmented DNA, the DNA can be digested with a methylation-dependent or methylation-sensitive restriction enzyme, intact DNA can be amplified using primers that hybridize to the adaptor sequences.
a second step can be performed to determine the presence, absence or quantity of a particular gene in an amplified pool of DNA.
the DNA is amplified using real-time, quantitative PCR.
the methods comprise quantifying the average methylation density in a target sequence within a population of genomic DNA.
the method comprises contacting genomic DNA with a methylation-dependent restriction enzyme or methylation-sensitive restriction enzyme under conditions that allow for at least some copies of potential restriction enzyme cleavage sites in the locus to remain uncleaved; quantifying intact copies of the locus; and comparing the quantity of amplified product to a control value representing the quantity of methylation of control DNA, thereby quantifying the average methylation density in the locus compared to the methylation density of the control DNA.
the quantity of methylation of a locus of DNA can be determined by providing a sample of genomic DNA comprising the locus, cleaving the DNA with a restriction enzyme that is either methylation-sensitive or methylation-dependent, and then quantifying the amount of intact DNA or quantifying the amount of cut DNA at the DNA locus of interest.
the amount of intact or cut DNA will depend on the initial amount of genomic DNA containing the locus, the amount of methylation in the locus, and the number (i.e., the fraction) of nucleotides in the locus that are methylated in the genomic DNA.
the amount of methylation in a DNA locus can be determined by comparing the quantity of intact DNA or cut DNA to a control value representing the quantity of intact DNA or cut DNA in a similarly-treated DNA sample.
the control value can represent a known or predicted number of methylated nucleotides.
the control value can represent the quantity of intact or cut DNA from the same locus in another (e.g., normal, non-diseased) cell or a second locus.
methylation density can refer to the number of methylated C-residues within a region.
the methylation-sensitive restriction enzyme is contacted to copies of a DNA locus under conditions that allow for at least some copies of potential restriction enzyme cleavage sites in the locus to remain uncleaved, then the remaining intact DNA will be directly proportional to the methylation density, and thus may be compared to a control to determine the relative methylation density of the locus in the sample.
a methylation-dependent restriction enzyme is contacted to copies of a DNA locus under conditions that allow for at least some copies of potential restriction enzyme cleavage sites in the locus to remain uncleaved, then the remaining intact DNA will be inversely proportional to the methylation density, and thus may be compared to a control to determine the relative methylation density of the locus in the sample.
Such assays are disclosed in, e.g., U.S. patent application Ser. No. 10/971,986.
Quantitative amplification methods can be used to quantify the amount of intact DNA within a locus flanked by amplification primers following restriction digestion.
Methods of quantitative amplification are disclosed in, e.g., U.S. Pat. Nos. 6,180,349; 6,033,854; and 5,972,602, as well as in, e.g., Gibson et al., Genome Research 6:995-1001 (1996); DeGraves, et al., Biotechniques 34(1):106-10, 112-5 (2003); Deiman B, et al., Mol. Biotechnol. 20(2):163-79 (2002). Amplifications may be monitored in “real time.”
Additional methods for detecting DNA methylation can involve genomic sequencing before and after treatment of the DNA with bisulfite. See, e.g., Frommer et al., Proc. Natl. Acad. Sci. USA 89:1827-1831 (1992). When sodium bisulfite is contacted to DNA, unmethylated cytosine is converted to uracil, while methylated cytosine is not modified.
restriction enzyme digestion of PCR products amplified from bisulfite-converted DNA is used to detect DNA methylation. See, e.g., Sadri & Hornsby, Nucl. Acids Res. 24:5058-5059 (1996); Xiong & Laird, Nucleic Acids Res. 25:2532-2534 (1997).
a MethyLight assay is used alone or in combination with other methods to detect DNA methylation (see, Eads et al., Cancer Res. 59:2302-2306 (1999)). Briefly, in the MethyLight process genomic DNA is converted in a sodium bisulfite reaction (the bisulfite process converts unmethylated cytosine residues to uracil). Amplification of a DNA sequence of interest is then performed using PCR primers that hybridize to CpG dinucleotides.
amplification can indicate methylation status of sequences where the primers hybridize.
the amplification product can be detected with a probe that specifically binds to a sequence resulting from bisulfite treatment of a unmethylated (or methylated) DNA. If desired, both primers and probes can be used to detect methylation status.
kits for use with MethyLight can include sodium bisulfite as well as primers or detectably-labeled probes (including but not limited to Taqman or molecular beacon probes) that distinguish between methylated and unmethylated DNA that have been treated with bisulfite.
kit components can include, e.g., reagents necessary for amplification of DNA including but not limited to, PCR buffers, deoxynucleotides; and a thermostable polymerase.
a Ms-SNuPE Metal-sensitive Single Nucleotide Primer Extension reaction
the Ms-SNuPE technique is a quantitative method for assessing methylation differences at specific CpG sites based on bisulfite treatment of DNA, followed by single-nucleotide primer extension (Gonzalgo & Jones, supra). Briefly, genomic DNA is reacted with sodium bisulfite to convert unmethylated cytosine to uracil while leaving 5-methylcytosine unchanged. Amplification of the desired target sequence is then performed using PCR primers specific for bisulfite-converted DNA, and the resulting product is isolated and used as a template for methylation analysis at the CpG site(s) of interest.
Typical reagents for Ms-SNuPE analysis can include, but are not limited to: PCR primers for specific gene (or methylation-altered DNA sequence or CpG island); optimized PCR buffers and deoxynucleotides; gel extraction kit; positive control primers; Ms-SNuPE primers for a specific gene; reaction buffer (for the Ms-SNuPE reaction); and detectably-labeled nucleotides.
bisulfite conversion reagents may include: DNA denaturation buffer; sulfonation buffer; DNA recovery regents or kit (e.g., precipitation, ultrafiltration, affinity column); desulfonation buffer; and DNA recovery components.
a methylation-specific PCR (“MSP”) reaction is used alone or in combination with other methods to detect DNA methylation.
An MSP assay entails initial modification of DNA by sodium bisulfite, converting all unmethylated, but not methylated, cytosines to uracil, and subsequent amplification with primers specific for methylated versus unmethylated DNA. See, Herman et al., Proc. Natl. Acad. Sci. USA 93:9821-9826, (1996); U.S. Pat. No. 5,786,146.
Additional methylation detection methods include, but are not limited to, methylated CpG island amplification (see, Toyota et al., Cancer Res. 59:2307-12 (1999)) and those described in, e.g., U.S. Patent Publication 2005/0069879; Rein, et al. Nucleic Acids Res. 26 (10): 2255-64 (1998); Olek, et al. Nat. Genet. 17(3): 275-6 (1997); and PCT Publication No. WO 00/70090.
Some embodiments provided herein include methods of determining the methylation status of a plurality of human nucleic acid loci. Some such embodiments include contacting a nucleic acid sample from a human subject with a reagent capable of providing an indication of the methylation status of said loci.
the loci comprise at least about 5 loci, at least about 10 loci, at least about 15 loci, at least about 20 loci, at least about 25 loci, at least about 50 loci, and at least about 100 loci.
the loci are selected from the group consisting of the loci listed in TABLE 6, TABLE 7, or TABLE 8.
the reagent is a restriction enzyme.
the reagent is a primer.
the reagent is a probe.
the reagent comprises sodium bisulfite.
Some embodiments provided herein relate to methods for identifying therapeutic agents.
Some such embodiments for identifying therapeutic agents which may be used to treat rheumatoid arthritis or osteoarthritis, can include contacting a cell with a test agent; and determining the methylation state of at least 1 locus selected from the loci listed in TABLE 6, TABLE 7, or TABLE 8 or at least one gene listed in Table 3 in the contacted cell.
the at least one locus may be selected from SEQ ID NO.s:1-485512.
Some methods also include comparing the methylation state of the at least 1 locus, or at least 1 gene in the contacted cell with the methylation state of the locus or the gene in the cell not contacted with the test agent, and selecting a test agent that increases or decreases the methylation state of the at least 1 locus or the at least 1 gene in the cell contacted with the test agent compared to the methylation state of the locus or the gene in a cell not contacted with the test agent. For example, if a locus, a gene group of loci or group of genes are hypermethylated in individuals with RA, agents which reduce the level of methylation at the locus, the gene, group of loci or group of genes may be useful as therapeutic agents.
agents which increase the level of methylation at the locus may be useful as therapeutic agents.
agents which produce a methylation profile in cells contacted with the agent having a greater similarity to the methylation profile of individuals who do not suffer from RA relative to the methylation profile in cells which have not been contacted with the agent may be useful as therapeutic agents.
test agents and potential therapeutic agents include small molecules (including but not limited to organic chemical compounds which have been obtained from natural sources or synthesized), nucleic acids (including but not limited to antisense nucleic acids, ribozymes, or siRNAs), peptides and proteins (including but not limited to cytokines TNF- ⁇ , and DMNTs).
nucleic acids including but not limited to antisense nucleic acids, ribozymes, or siRNAs
peptides and proteins including but not limited to cytokines TNF- ⁇ , and DMNTs.
At least 1 locus is selected from the group consisting of the loci listed in TABLE 6. In some embodiments, at least 1 locus is selected from the group consisting of the loci listed in TABLE 7. In some embodiments, the at least one locus may be selected from SEQ ID NO.s:1-485512. In some embodiments, at least 1 gene is selected from the group consisting of the gene listed in TABLE 3.
the methylation states of at least about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 loci selected from the group consisting of the loci listed in TABLE 6, TABLE 7, or TABLE 8 are determined. In some embodiments, the methylation states of at least about 5, 10, 20, 30, 40, 50, 60, 70, 80, or 100 loci selected from the group consisting of the loci of SEQ ID NO.s:1-485512. In some embodiments, the methylation states of at least about 1, 5, 10, 20, 30, 40, 50 genes selected from the group consisting of the genes listed in TABLE 3 are determined.
the cell comprises a synoviocyte, such as a fibroblast-like synoviocyte, for example, a rheumatoid arthritis fibroblast-like synoviocyte or an osteoarthritis fibroblast-like synoviocyte.
the cell comprises a macrophage.
the cell comprises a peripheral blood cell.
the peripheral blood cell is selected from the group consisting of white blood cell, including neutrophil, eosinophil, basophil, lymphocyte, B cell, plasma cell, T cell, natural killer cell, monocyte, and dendritic cell.
the cell is mammalian, e.g., human.
More embodiments of methods for identifying therapeutic reagents include identifying agents that modulate methylation of genes encoding proteins that act in the same pathway as other proteins encoded by genes that are differentially methylated in rheumatoid arthritis or osteoarthritis or agents which modulate the activity of proteins in the same pathway as proteins encoded by genes which are differentially methylated in rheumatoid arthritis or osteoarthritis.
Some methods for identifying a therapeutic agent for treating rheumatoid arthritis or osteoarthritis include contacting a cell with a test agent, and determining the methylation state of at least 1 gene selected from a gene encoding a protein that acts in a pathway that includes a protein encoded by a gene that is differentially methylated in a rheumatoid arthritis cell or osteoarthritis cell compared to a normal cell.
Some methods also include comparing the methylation state of the at least 1 gene in the contacted cell with the methylation state of the gene in a cell which was not contacted with the test agent, selecting a test agent that increases or decreases the extent of methylation of the at least 1 gene in the cell contacted with the test agent compared to the extent of methylation of the at least 1 gene in a cell which was not contacted with the test agent such that the extent of methylation of the at least 1 gene in the cell contacted with the test agent is a methylation state associated with the absence of rheumatoid arthritis or osteoarthritis or with a reduction in the symptoms associated with rheumatoid arthritis or osteoarthrities.
the pathway is selected from focal adhesion, glycosphingolipid biosynthesis—lacto and neolacto series, arrhythmogenic right ventricular cardiomyopathy (ARVC), ECM-receptor interaction, amoebiasis, leukocyte transendothelial migration, protein digestion and absorption, cell adhesion molecules (CAMs), nitrogen metabolism, ErbB signaling pathway, African trypanosomiasis, primary bile acid biosynthesis, Fc epsilon RI signaling pathway, mTOR signaling pathway, and adipocytokine signaling pathway.
ARVC arrhythmogenic right ventricular cardiomyopathy
ECM-receptor interaction amoebiasis
leukocyte transendothelial migration protein digestion and absorption
CAMs cell adhesion molecules
nitrogen metabolism ErbB signaling pathway
African trypanosomiasis African trypanosomiasis
primary bile acid biosynthesis Fc epsilon RI signaling pathway
kits can be useful for diagnosing, determining a prognosis, or determining a response to treatment of a subject with a disorder, such as rheumatoid arthritis, comprising: a reagent for determining the methylation state of at least one locus selected from the group consisting of the loci listed in TABLE 6, TABLE 7, or TABLE 8.
the at least one locus may be selected from SEQ ID NO.s:1-485512.
the kit also includes at least one polynucleotide primer comprising a sequence hybridizing to at least a portion of the at least one locus selected from the group consisting of the loci listed in TABLE 6, TABLE 7, or TABLE 8.
the locus may be selected from SEQ ID NO.s:1-485512.
the kit can include one or more of methylation-dependent restriction enzymes, methylation-sensitive restriction enzymes, amplification (e.g., PCR) reagents, probes and/or primers.
a reagent can determine the methylation states of at least about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more than 100 loci selected from the group consisting of the loci listed in TABLE 6, TABLE 7, or TABLE 8.
the at least about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more than 100 loci may be selected from SEQ ID NO.s:1-485512.
kits for diagnosing, determining a prognosis, or determining or predicting a response to treatment of a subject with rheumatoid arthritis or osteoarthritis comprising a reagent for determining the methylation state of at least one gene selected from the group consisting of the genes listed in TABLE 3.
kits also include at least one polynucleotide primer comprising a sequence hybridizing to at least a portion of the at least one gene selected from the group consisting of the genes listed in TABLE 3.
the reagent comprises a restriction enzyme.
a reagent can determine the methylation states of at least about 1, 5, 10, 20, 30, 40, 50, or more genes selected from the group consisting of the genes listed in TABLE 3.
nucleic acid array consisting essentially of nucleic acids useful for diagnosing rheumatoid arthritis or osteoarthritis, determining a prognosis of rheumatoid arthritis or osteoarthritis, or determining or predicting a response to treatment of a subject being evaluated for or suffering from rheumatoid arthritis or osteoarthritis.
the nucleic acids comprise at least about 5 loci, at least about 10 loci, at least about 15 loci, at least about 20 loci, at least about 25 loci, at least about 50 loci, and at least about 100 loci.
the loci are selected from the group consisting of the loci listed in TABLE 6, TABLE 7, or TABLE 8.
the loci may be selected from SEQ ID NO.s:1-485512.
the calculations for the methods described herein can involve computer-based calculations and tools. For example, a methylation value for a DNA region or portion thereof can be compared by a computer to a threshold value, as described herein.
the tools are advantageously provided in the form of computer programs that are executable by a general purpose computer system (referred to herein as a “host computer”) of conventional design.
the host computer may be configured with many different hardware components and can be made in many dimensions and styles (e.g., desktop PC, laptop, tablet PC, handheld computer, server, workstation, mainframe). Standard components, such as monitors, keyboards, disk drives, CD and/or DVD drives, and the like, may be included.
the connections may be provided via any suitable transport media (e.g., wired, optical, and/or wireless media) and any suitable communication protocol (e.g., TCP/IP); the host computer may include suitable networking hardware (e.g., modem, Ethernet card, WiFi card).
suitable transport media e.g., wired, optical, and/or wireless media
TCP/IP any suitable communication protocol
the host computer may include suitable networking hardware (e.g., modem, Ethernet card, WiFi card).
the host computer may implement any of a variety of operating systems, including UNIX, Linux, Microsoft Windows, MacOS, or any other operating system.
Computer code for implementing aspects of the present invention may be written in a variety of languages, including PERL, C, C++, Java, JavaScript, VBScript, AWK, or any other scripting or programming language that can be executed on the host computer or that can be compiled to execute on the host computer. Code may also be written or distributed in low level languages such as assembler languages or machine languages.
the host computer system advantageously provides an interface via which the user controls operation of the tools.
software tools are implemented as scripts (e.g., using PERL), execution of which can be initiated by a user from a standard command line interface of an operating system such as Linux or UNIX.
commands can be adapted to the operating system as appropriate.
a graphical user interface may be provided, allowing the user to control operations using a pointing device.
the present invention is not limited to any particular user interface.
Scripts or programs incorporating various features of the present invention may be encoded on various computer readable media for storage and/or transmission.
suitable media include magnetic disk or tape, optical storage media such as compact disk (CD) or DVD (digital versatile disk), flash memory, and carrier signals adapted for transmission via wired, optical, and/or wireless networks conforming to a variety of protocols, including the Internet.
Some embodiments include methods for diagnosing rheumatoid arthritis or osteoarthritis, determining a rheumatoid arthritis or osteoarthritis prognosis, or determining or predicting a response to treatment for rheumatoid arthritis or osteoarthritis in a subject comprising accessing first data representing nucleic acid loci which are differentially methylated in individuals with rheumatoid arthritis, osteoarthritis a rheumatoid arthritis or osteoarthritis prognosis, or a response to treatment for rheumatoid arthritis or osteoarthritis wherein said data is stored on a non-transitory computer readable medium.
Some embodiments also include instructing a computer to compare said first data to second data representing the methylation status of said nucleic acid loci in a sample taken from said subject, wherein said data representing the methylation status of said nucleic acid loci in a sample taken from said subject is stored on a non-transitory computer readable medium.
Some embodiments also include diagnosing rheumatoid arthritis or osteoarthritis, determining a rheumatoid arthritis or osteoarthritis prognosis, or determining or predicting a response to treatment for rheumatoid arthritis or osteoarthritis in said subject if said first data representing the methylation status of said nucleic acid loci in a sample taken from said subject are significantly similar to said second data representing nucleic acid loci which are differentially methylated in individuals with rheumatoid arthritis, osteoarthritis a rheumatoid arthritis or osteoarthritis prognosis, or a response to treatment for rheumatoid arthritis or osteoarthritis.
Synovial tissues were minced and incubated with 0.5 mg/ml collagenase VIII (Sigma) in serum-free RPMI (Mediatech, VA) for 1.5 h at 37° C., filtered through a 0.22 ⁇ m cell strainer, extensively washed, and cultured in DMEM supplemented with 10% FCS (endotoxin content ⁇ 0.006 ng/ml; Gemini Biosciences, CA), penicillin, streptomycin, gentamicin and L-glutamine in a humidified 5% CO 2 incubator. After overnight culture, nonadherent cells were removed, and adherent cells were trypsinized, split at a 1:3 ratio, and cultured. Synoviocytes were used from passage 4 through 9, when FLS were a homogeneous population with ⁇ 1% CD11b, ⁇ 1% phagocytic, and ⁇ 1% FcR II positive cells.
Synovial tissues were obtained at the time of clinically indicated total knee or hip joint replacement surgery except for one patient with RA who had wrist surgery.
the mean ages of RA and OA patients were 53 ⁇ 9 and 68 ⁇ 16, respectively. Additional information on four patients (2 RA and 2 OA) was limited because the samples were de-identified.
the erythrocyte sedimentation rates for the remaining RA and OA patients were 38 ⁇ 15 and 19 ⁇ 10, respectively.
3 were seropositive for serum rheumatoid factor or anti-CCP antibody and all were treated with low dose prednisone, 2 with methotrexate, 2 with a TNF blocker, and 1 with leflunomide.
OA was mainly treated with acetaminophin and narcotics for pain.
RA and OA FLS were grown to 80% confluence and harvested.
Genomic DNA of 10 6 FLS was isolated using the MagMAXTM DNA Multi-Sample Kit (Applied Biosystems). DNA quality and quantity was assessed with a NanoDrop ND-2000 spectrometer (NanoDrop Technologies, Wilmington, Del., USA).
mRNA from cultured FLS was isolated using RNA-STAT (Tel-Stat, TX) and cDNA was prepared, according to manufacturer's instructions using GeneAmp 2400 (Applied Biosystems).
Quantitative real-time PCR was performed using Assays On Demand (Applied Biosystems) to determine relative mRNA levels using the GeneAmp 5700 Sequence Detection System (Applied Biosystems) as described previously. Standard curves for human MMP1 and GAPDH were generated. Sample Ct values were used to calculate the number of cell equivalents in the test samples. The data were then normalized to GAPDH expression to obtain relative cell equivalents.
Genomic DNA was isolated from female RA and OA FLS as described.
the Infinium HumanMethylation450 chip was processed as described by the manufacturer (Illumina, San Diego, Calif.). This chip covers 96% of RefSeq genes and provides comprehensive gene region coverage, targeting multiple sites with promoter, 5 ⁇ UTR, 1st exon, gene body and 3 ⁇ UTR.
Initial analysis was performed with the Genome Studio methylation module, and then further analysed as described herein. The methylation level of a loci is measured as:
M is the fluorescence level of the methylation probe and U is the methylation level of the unmethylated probe.
a constant value of 100 is added to prevent division by a small number (or 0) when background subtraction was used.
the ⁇ values varied from 0 (completely unmethylated) to 1 (completely methylated). To measure the difference in methylation at a loci between OA and RA the average ⁇ levels were compared.
EF is greater than 1 then it means the genes loci are enriched with differentially methylated loci.
a P-value for the level of enrichment was calculated using the hypergeometric distribution. The resulting P-values were corrected with the Benjamini-Hochberg correction. Genes with enrichment P-values beneath ⁇ 0.05 were recorded. If a gene was found to be enriched at multiple loci differentially methylated levels then only the level with the lower enrichment P-value was reported.
Pathway enrichment was carried out using the KEGG human pathways and modules (www.genome.jp/kegg/download). The enrichment analysis of 1859 loci was performed by mapping pathway to loci via the loci gene annotations, the EF of loci being enriched in KEGG pathway as calculated. If EF was greater than 1 then P-value for the level of enrichment was calculated using the hypergeometric distribution. The resulting P-values were corrected with the Benjamini-Hochberg correction. As the KEGG pathways represent groups of related bimolecular pathways a P-value cut-off of ⁇ 0.1 was used as it would allow enrichment within individual bimolecular pathways to be identified.
GO term enrichment analysis was carried out using model-based gene set analysis which uses probabilistic inference to identify the active GO terms (Bauer, et al. 2010 GOing Bayesian: model-based gene set analysis of genome-scale data. Nucleic Acids Res. 2010; 38:3523-32, incorporated by reference in its entirety). This approach naturally deals with overlapping GO categories and avoids the need for multiple testing correction. Marginal probability values>0.50 were considered significantly enriched.
the DNA methylome in RA and control (OA) FLS was evaluated.
the Infinium HumanMethylation450 chip (Illumina, Inc.) was used to determine the methylation status of 485,512 loci from FLS isolated from 11 female patients at the time of total joint replacement surgery (6 RA; 5 OA). Loci were removed from subsequent analysis if any of the probes for a locus could not be disguised from background with a P-value ⁇ 0.01 or if enough beads present upon the chip for accurate measurement of their methylation level. After filtering, 476,331 loci were available for further analysis.
RA and OA FLS To assess global methylation status of RA and OA FLS, the methylation scores over all filtered loci within a sample were summed. The difference between the two samples was assessed using Student's t-test. Initial analysis included all loci and was then repeated for only loci located in promoters. There were no significant differences between RA and OA (P-values 0.528 and 0.627, respectively). Therefore, globally hypo- or hypermethylation is not associated with RA when compared to OA.
genes identified with statistically significant differences between RA and OA at an individual locus using the ILLUMINA dataset are shown in TABLE 2.
CpG methylation was significantly different in a number of genes implicated in RA. Several genes implicated in inflamation and immune responses are differentially methylated in RA.
TABLE 6 and TABLE 7 list loci which are differentially methylated in RA compared to OA; a positive OA-RA value represents a loci which is hypomethylated in RA FLS (TABLE 6), a negative OA-RA value represents a loci which is hypermethylated in RA FLS (TABLE 7).
Permutation analysis was carried out to assess the significance of loci identified as differentially methylated.
the 11 samples were randomly assigned to OA and RA while maintaining the same number of OA and RA labels, i.e., 5 OA and 6 RA.
the permutation analysis was repeated 1,000 times.
the average number of significant loci during the permutation analysis was 4.9, compared to 1,859 for the correct disease identification.
the permutation analysis strongly supports these loci as truly differentially methylated and not as a result of random chance.
the methylation patterns of the loci across the 11 samples were hierarchically clustered ( FIG. 2 ).
the clustering of the samples distinguishes OA from RA, which clearly segregate according to disease type.
the clustering of the loci also shows that groups of loci have similar patterns of differentially methylated across the samples.
FIG. 4 depicts the pathway ‘Focal adhesion’, which was enriched with loci that are significantly less methylated in RA.
a list of differential GO terms and an analysis of hypomethylated and hypermethylated terms in RA with marginal probability>0.5 is shown in TABLE 4.
hypomethylated DNA in RA was greater than for hypermethylated DNA (15 vs. 3).
Cytoscape was used to evaluate networks affected by differential methylation and to determine likely targets for subsequent analysis.
interactions between hypomethylated genes and their neighbors were evaluated.
a portion of the Cytoscape analysis is shown in FIG. 5 .
Significant nodes included key hypomethylated loci (e.g., STAT3, MAP3K5, CHI3L1, STK24) (circle size indicates degree of hypomethylation).
FIG. 5 depicts how these loci interact with each other and with multiple additional pathways, and describes how the methylated genes might regulate inflammatory responses in RA. This map shows only a fraction of the interactions and provides an unbiased view of how methylation affects synoviocyte function.
FIG. 6 depicts graphs of relative expression of DNMT1, DNMT3a, and DNMT3b in OA FLS and RA FLS. Unexpectedly, expression levels of DNMTs in RA and OA were found to be similar. In view of these findings, changes in DNMT expression in FLS was examined, and the role of cytokines, e.g., IL-1, in RA, that may contribute to a DNMT profile was examined.
cytokines e.g., IL-1
IL-1 Decreases DNMT Function in FLS
a functional assay was performed to determine if IL-1 suppresses DNA methylation activity of the DNMTs.
FLS were stimulated with 1 ng/ml of IL-1 for 14 days and extracts were assayed using the DNMT Activity/Inhibition Assay (Active Motif Co., Carlsbad, Calif.), which is an ELISA-based method that measures methylation of a CpG-enriched DNA substrate.
DNMT Activity/Inhibition Assay Active Motif Co., Carlsbad, Calif.
FLS form the synovial intimal lining and play an integral role in the pathogenesis of RA by producing key cytokines, small molecule mediators, and proteases. While osteoclasts are the primary effectors of bone erosions in arthritis, FLS are responsible for cartilage damage by virtue of their ability to adhere to and invade the cartilage extracellular matrix. This capacity requires homotypic aggregation mediated by the adhesion molecule cadherin-11, which directs intimal lining formation and supports an invasive phenotype. Understanding the molecular mechanisms that regulate FLS activation could provide insights into the pathogenesis of RA and lead to novel therapeutic strategies. In the present application, the epigenetic profile of RA was evaluated by exploring a newly discovered DNA methylation signature that could potentially affect adaptive and innate immune functions, through their effects on synoviocytes and immune cells in the blood and joint.
Rheumatoid FLS exhibit a unique aggressive phenotype that contributes to the cytokine milieu and joint destruction. Functional studies suggest that RA cells are imprinted in situ and maintain these features after many passages in tissue culture. For example, RA FLS, unlike OA or normal synoviocytes, adhere to and invade cartilage explants in SCID mice. RA FLS can grow under anchorage-independent conditions, are less susceptible to contact inhibition, resistant to apoptosis. RA synoviocytes can potentially “metastasize” and transfer the invasive phenotype from one joint to another.
DNA methylation could be especially relevant in RA, in light of role in neoplasia and embryonic growth.
Normal ontogeny relies on a carefully orchestrated sequence of DNA methylation to repress regulatory genes by methylating cytosine in CpG loci, either in promoters or in genes themselves.
Methylation abnormalities have been associated with many diseases, most notably cancer where renewed expression or inappropriate suppression of genes allows cells to escape normal homeostatic controls. Hypomethylation and hypermethylation are associated with many malignancies and can contribute to transformation.
DNA methyltransferases are responsible for initiating and maintaining CpG methylation in the human genome by converting cytosine to methylcytosine.
DNMT1, DNMT3a, and DNMT3b are the primary enzymes responsible for CpG methylation.
DNMT3a and DNMT3b mainly regulate de novo methylation while DNMT1 maintains methylation, especially during cell division.
DNMT expression and DNA methylation are not immutable but are influenced by the environment and modify gene expression throughout life and even in progeny. For instance, pregnant mice fed a diet rich in methyl donors give birth to pups with increased levels of DNA methylation and increased airway reactivity in murine asthma for at least two subsequent generations.
RA FLS Global hypomethylation has been described in peripheral blood mononuclear cells of patients with RA, although the specific genes involved are not known. Modest global hypomethylation was also reported in cultured RA FLS when compared to OA cells. In contrast, our studies showed that global methylation levels are similar in OA and RA FLS using two different techniques (ELISA-based and chip based). Thus, RA FLS do not appear to be hypomethylated overall, but like neoplasia, display a pattern of hypermethylated and hypomethylated genes.
the ILLUMINA chip analysis identified distinct methylation profiles of OA and RA FLS involving 1859 loci located in 1206 genes. Cluster analysis showed that the two types of FLS could be easily distinguished based solely on the methylation patters. The results were confirmed using a variety of additional analyses that reduced the possibility of random chance as an explanation. Additional analysis identified 207 genes with multiple hyper- or hypomethylated loci. Many of these genes play a key role in inflammation, matrix regulation, leukocyte recruitment and immune responses. Gene expression levels correlated with methylation status, with high expression in hypomethylated genes in RA FLS and normal expression in genes that were not differentially methylated. Given the number of influences that can potentially alter gene expression in vitro, the general concordance between methylation and mRNA levels was striking.
the data described herein provide evidence that epigenetic changes are present in RA synoviocytes and that they persist in culture. Imprinting could potentially occur before clinical disease and contribute to susceptibility. Alternatively, and perhaps more likely, the changes can be induced after initiation of synovitis. In the latter situation, the inflammatory milieu could potentially imprint synoviocytes and affect their function for many passages. Thus, local inflammation could potentially alter expression of enzymes responsible for initiating and maintaining DNA methylation. This process imprints synoviocytes, peripheral blood cells that are present in synovium for a short period of time, and immune cells, alters their behaviour, and ultimately changes the natural history of disease.
RA and control cells could provide major insights into how the epigenetic profile of various tissues contribute to the pathogenesis of RA.
it could also lead to interesting diagnostic or personalized medicine applications after sufficient data are available to correlate the methylome to phenotype.
DB CPT tube (BD cat#362760) and inverted 10 times. Four tubes per patient were drawn. The tubes were stored at room temperature until processed. Processing was performed in less than four hours after being drawn. The tubes were spun 30 minutes at 3000 rpm. The mononuclear cell layer was carefully removed and washed in at least 10 volumes of cold PBS with 0.1% BSA (Buffer 1: PBS Invitrogen cat#14190, BSA Gemini cat#700-100P). Mononuclear cells were then spun at 1600 rpm for 10 minutes.
BSA Buffer 1: PBS Invitrogen cat#14190, BSA Gemini cat#700-100P
Cells were then resuspended in 3 ml of cold PBS supplemented with 0.1% BSA and 2 mM EDTA (Buffer 2: Buffer 1 plus EDTA Invitrogen cat#15575020). Cells were then counted and divided into 2 ml eppendorf tubes as follows: 0.5 ml for T-cell isolation, 1.5 ml for B-cell isolation, 0.75 ml for monocyte isolation, and 0.25 ml for whole PBMCs. These were spun again at 1600 rpm for 10 minutes and supernatants were discarded. The whole PBMC fraction was frozen while the others continued the specific cell type separation.
Dynabeads magnetic beads (CD19 pan B Invitrogen cat#111-43D, CD2 pan T Invitrogen cat#111-59D, CD14 monocyte Invitrogen cat#111-49D) were used for the specific cell-type isolation.
the magnetic bead mixtures were added to 1 ml of cold Buffer 2 according to the number of cells (50 ⁇ l T-cell bead isolation mixture per 10 7 cells, 25 ⁇ l B-cell bead isolation mixture per 2.5 ⁇ 10 7 cells, and 25 ⁇ l monocyte isolation bead mixture per 10 7 cells), mixed well and applied to the magnet for 3 minutes to wash the beads.
the protocol for cultured cells was followed and included the recommendation of RNase A (Qiagen cat#19101) treatment.
DNA eluents were then concentrated using Amicon ultra 30K filers (Millipore cat# UFC503096). Concentrated DNA was then quantified using Quant-it Picogreen reagent (Invitrogen cat# P7589). Concentrations were then standardized to 100 ng/ ⁇ l.
PBMC preparations may also be separated into other cell types, such as white blood cells, neutrophils, eosinophils, basophils, lymphocytes, plasma cells, natural killer cells, and dendritic cells using procedures such as those described above or other methods familiar to those skilled in the art.
Macrophages may be separated from other cell types using methods well known in the art.
particular cell types can be enriched and/or isolated using a variety of methods, such methods are well known in the art and include immunological methods, fluorescent activated cell sorting (FACS) methods, and affinity chromatography methods.
FACS fluorescent activated cell sorting
cells such as eosinophils can be enriched/isolated using antibodies specific to specific receptors on the cell surface such as L-selectin, and VLA-4 (Sriramarao P., et al., (1994) J. Immunol. 153:4238-46, incorporated by reference in its entirety)
Neutrophils may be isolated using density gradients or using antibodies specific to other cell surface receptors (Firestein G. S., et al., (1995) J. Immunol. 154:326-34, incorporated by reference in its entirety).
Samples of genomic DNA are obtained from peripheral blood mononuclear cells or specific cell types separated from PBMC preparations as described above.
the samples are obtained from individuals with RA, individuals with OA, individuals with a known prognosis for rheumatoid arthritis or osteoarthritis, individuals with a known reponse to treatment for rheumatoid arthritis or osteoarthritis and control subjects without rheumatoid arthritis, without osteoarthritis, without the known prognosis for rheumatoid arthritis or osteoarthritis, or without the known response to treatment for rheumatoid arthritis or osteoarthritis using methods described herein.
the methylation states of loci for each set of genomic DNA is determined as described herein for genomic DNA from FLS cells.
hypomethylated and hypermethylated loci in individuals with RA, individuals with OA, individuals with a known prognosis for rheumatoid arthritis or osteoarthritis individuals with a known response to treatment for rheumatoid arthritis or osteoarthritis are identified by comparing the methylation states of the loci to the methylation states of the loci in control subjects without rheumatoid arthritis, without osteoarthritis, without the known prognosis for rheumatoid arthritis or osteoarthritis, or without the known response to treatment for rheumatoid arthritis or osteoarthritis respectively using the methods described herein for FLS cells.
hypomethylated and hypermethylated loci in RA genomic DNA relative to the methylation state of loci in OA genomic DNA are identified. In other embodiments hypomethylated and hypermethylated loci in RA genomic DNA relative to the methylation state of loci in genomic DNA from control subjects without RA are identified. In some embodiments, hypomethylated and hypermethylated loci in OA genomic DNA relative to the methylation state of loci in genomic DNA are identified from control subjects without OA are identified.
Genes with multiple differentially methylated loci are analyzed as described herein for genomic DNA from FLs cells, Gene expression and methylation status are analyzed as described herein for genomic DNA from FLs cells, Pathway analyses and gene ontology analyses are performed as described herein for genomic DNA from FLs cells. Network and DNMT analyses as described herein for genomic DNA from FLs cells.
Genomic DNA is obtained from a subject and the methylation states of one or more loci having differential methylation in individuals with individuals with RA, individuals with OA, individuals with a particular prognosis for rheumatoid arthritis or osteoarthritis, or individuals with a particular response to treatment for rheumatoid arthritis or osteoarthritis is determined.
the methylation state of the one or more differentially methylated loci in the genomic DNA from the subject is compared with the methylation state of the one or more differentially methylated loci in normal tissue, tissue from a subject with a known prognosis, or tissue from a subject with a known response to treatment.
the methylation state of the one or more differentially methylated loci in the genomic DNA from the subject is compared with a methylation state of the one or more differentially methylated loci known to be indicative of RA or a lack thereof, OA or a lack thereof, a particular prognosis for rheumatoid arthritis or osteoarthritis or a lack thereof, or a particular response to treatment for rheumatoid arthritis or osteoarthritis or a lack thereof.
the genomic DNA may be obtained from any desired cell type, including the cell types listed herein.
the genomic DNA may be obtained from FLS cells, a peripheral blood sample or a specific cell type separated from a PBMC sample obtained from a subject. If the methylation state of the one or more differentially methylated loci is a methylation state known to be indicative of RA, a particular prognosis for rheumatoid arthritis or osteoarthritis, or a particular response to treatment for rheumatoid arthritis or osteoarthritis the subject is determined to have RA, the particular prognosis for rheumatoid arthritis or osteoarthritis, or the particular response to treatment for rheumatoid arthritis or osteoarthritis. A treatment regimen consistent with this determination may then be administered.
Genomic DNA is obtained from a subject without RA and a subject without OA and the methylation states of one or more loci having differential methylation in RA FLS and OA FLS cells, respectively, is determined.
the methylation state of the one or more differentially methylated loci in RA FLS and OA FLS cells is compared to a methylation state of the one or more differentially methylated loci in the genomic DNA from a subject without RA and in the genomic DNA from a subject without OA, respectively.
the methylation state of loci indicative of the absence of RA or OA in a subject are determined.
the genomic DNA may be obtained from any desired cell type, including the cell types listed herein.
the genomic DNA may be obtained from FLS cells, a peripheral blood sample or a specific cell type separated from a PBMC sample obtained from a subject. If the methylation state of the one or more differentially methylated loci is a methylation state known to be indicative of RA, a particular prognosis for rheumatoid arthritis or osteoarthritis, or a particular response to treatment for rheumatoid arthritis or osteoarthritis the subject is determined to have RA, the particular prognosis for rheumatoid arthritis or osteoarthritis, or the particular response to treatment for rheumatoid arthritis or osteoarthritis. A treatment regimen consistent with this determination may then be administered.
PBMCs Peripheral Blood Mononuclear Cells
methylation states of loci in PBMCs were determined with methods substantially similar to those described herein using the ILLUMINA HumanMethylation 450 BeadChip.
Differentially methylated loci (DML) were identified with average methylation differences between OA and RA of >0.10.
RA/OA differential methylation was conducted using a t-test. P-values were converted to q-values to account for multiple hypothesis testing, and DML with q-values ⁇ 0.25 were considered to be potential PBMC biomarkers.
TABLE 8 lists about 2544 RA/OA PBMC DML with associated data including the OA-RA average methylation difference, CG identifier associated with the Illumina 450K beadchip (Locus), associated genes, and the genomic location of the potentially methylated C of the CpG (+ strand) determined using the UCSC hg19 reference genome.
the CpG* column of Table 8 lists values for “chromosome:chromosome co-ordinate.”

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WO2013033627A2 (fr)	2013-03-07
WO2013033627A3 (fr)	2013-05-02

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